Journal of Cell Science 105, 359-367 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
359
Centromeric DNA cloned from functional kinetochore fragments in mitotic
cells with unreplicated genomes
Ilia I. Ouspenski and B. R. Brinkley*
Department of Cell Biology, Baylor College of Medicine, Houston, TX, 77030, USA
*Author for correspondence
SUMMARY
Treatment of cells arrested in the cell cycle at the G1/Sphase boundary with 5 mM caffeine induces premature
mitosis, resulting in chromosomal fragmentation and
detachment of centromere-kinetochore fragments,
which are subsequently attached to the mitotic spindle
and segregated in anaphase. Taking advantage of this
in vivo separation of the centromere, we have developed
a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have
isolated and cloned DNA from the centromere-enriched
material of Chinese hamster cells. One of the clones thus
obtained was characterized in detail. It contains 6 kb of
centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric
sequences and is devoid of any extensive repetitive struc-
ture. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of
the clone include the presence of several A+T-rich
regions and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We
hypothesize that these features might be related to the
more compact packaging of centromeric chromatin in
interphase nuclei and mitotic chromosomes.
INTRODUCTION
identified in the centromere region of higher eukaryotic
chromosomes. In humans, these include various copy numbers of alpha and beta satellites and three types of ‘classic’
satellites. Moreover, some satellite sequences have features
that suggest their functional significance in centromere
organization. For example, many copies of the alpha satellite repeats contain the consensus binding sequences for the
centromere protein CENP-B (known as the CENP-B box)
(Willard and Waye, 1987; Masumoto et al., 1989a). CENPB is present at or near the kinetochore and may be important for mitotic segregation (Cook et al., 1990; Bernat et
al., 1990; Simerly et al., 1990). When introduced into an
African green monkey cell line, human alpha satellite DNA
was associated with abnormal mitotic chromosome segregation, suggesting a specific role in centromere function
(Haaf et al., 1992). A pentameric repetitive sequence that
is a prominent component of classic satellites II and III,
was found to be conserved in many species ranging from
mammals to Drosophila, indicating a potentially significant
function (Grady et al., 1992). In the mouse genome, two
classes of repeats are known to be located in the centromere
region, major and minor satellites. The minor satellite might
be functionally equivalent to alphoid DNA in the human
genome since it also contains the CENP-B box sequence
(Wong and Rattner, 1988). Another family of simple
The centromere is a complex segment of the eukaryotic
chromosome defined by a discrete primary constriction
flanked on both sides by a pair of sister kinetochores. It
functions to achieve the pairing of sister chromatids and
segregation of chromosomes in mitosis and meiosis. Studies
of the chromosomes of budding yeast (reviewed by Clarke
and Carbon, 1985) indicate that proper segregation leading
to mitotic stability is conferred by an A+T-rich segment of
DNA approximately 125 bp long. The search for similar
functional sequences in centromeres of other eukaryotes has
been complicated by their substantially larger size and the
presence of blocks of heterochromatin containing repetitive
sequences. For example, the centromere of fission yeast
spans over 80 kb of non-transcribed DNA and, like centromeres of higher eukaryotes, contains several classes of
repetitive sequences. The minimal region of the centromere
sequence that confers mitotic stability to minichromosomes
is over 40 kb long and contains four classes of repeats as
well as a central core element (Clarke and Baum, 1990).
Moreover, segregation of minichromosomes in meiosis I
requires additional centromere-specific sequences, indicating the complexity of this region.
Several types of repetitive DNA sequences have been
Abbreviations: MUG, mitotic cells with unreplicated genomes;
MAR, matrix associated regions
Key words: centromere, kinetochore, mitosis, chromosome
segregation, in situ hybridization
360
I. I. Ouspenski and B. R. Brinkley
sequence centromeric repeats, dodeca satellite, was isolated
from the Drosophila genome (Abad et al., 1992). It was
found to be highly conserved and located at some human
centromeres.
Although progress has been made in identifying and
characterizing DNA in the centromere region of higher
eukaryotic chromosomes, specific sequences conferring
centromeric function remain largely unknown. In order to
understand the molecular basis of chromosome segregation
in mitosis and meiosis, as well as possible causes of mitotic
errors leading to aneuploidy, functionally significant DNA
sequences must be identified. In this report we present a
novel approach for isolating and cloning sequences from
functional subfragments of the centromeres of mammalian
chromosomes. The rationale for the procedure is based on
the induction of cells with unreplicated genomes to go into
mitosis prematurely. This is achieved by sequential treatment with hydroxyurea and caffeine as described initially
by Schlegel and Pardee (1986). Such treatment results in
the fragmentation and detachment of centromere-kinetochore fragments from chromosomes and their subsequent
attachment to the mitotic spindle and segregation in
anaphase (Brinkley et al., 1988; Zinkowski et al., 1991).
Here we report the procedure for isolation of DNA from a
chromatin fraction enriched in kinetochore fragments of
MUGs and describe the cloning of centromere-specific
sequences from chromosome 1 of Chinese hamster cells.
MATERIALS AND METHODS
Indirect immunofluorescence staining of cells
This was performed as described previously (Zinkowski et al.,
1991). Autoimmune antiserum SH of a patient with CREST scleroderma was used at 1:300 to 1:1000 dilution; the set of antigens
recognized by this serum in CHO cells is described in the text.
For immunofluorescence analysis of fractionated cell lysate, samples were diluted with the buffer AS (described below) to reduce
the concentration of glycerol or Percoll to 10% or lower, and spun
onto glass microscope slides in a Cytospin-3 cytocentrifuge (Shandon) at 1000 r.p.m. for 5 min. The specimens were fixed in 3%
formaldehyde in buffer AS without digitonin, then washed with
PBS and treated for immunofluorescence staining in the same way
as whole cells.
Electron microscopy
Electron microscopy was performed according to previously published protocols (Zinkowski et al., 1991).
Electrophoresis
This was performed using the buffer system of Laemmli (1970)
in a 10% polyacrylamide gel. Prestained marker proteins of 30120 kDa (Sigma) were used for the determination of the relative
molecular mass of the proteins. Fractions of glycerol and Percoll
gradients were diluted with AS buffer and concentrated using Centricon-10 microconcentrators (Amicon) to reduce the concentrations of glycerol or Percoll to 10% or lower, and mixed with an
equal volume of 2 × sample buffer (Laemmli, 1970). Immunoblotting and dot-blot analyses were carried out following Towbin and
Gordon (1984) using antiserum SH diluted 1:500 followed by
alkaline phosphatase-conjugated goat antibodies to human IgG
(Boehringer Mannheim). The antigen-antibody complexes were
visualized using the NBT/BCIP substrate pair. Protein concentra-
tion was determined by the method of Schaffner and Weissmann
(1973).
Generation of mitotic cells with unreplicated
genomes
This was done as previously described (Zinkowski et al., 1991).
Chinese hamster ovary cell line CHO-K1 was used throughout the
study. Cells were cultured in McCoy’s 5A medium supplemented
with 2.5% fetal calf serum and 10% calf serum in a 5% CO2 incubator. Cells were grown to confluence and subcultured to 20%
confluence into the medium containing 2 mM hydroxyurea.
Twenty hours later, caffeine and colcemid were added to final
concentrations of 5 mM and 0.15 mg/ml, respectively. Mitotic
cells were collected by shake-off 8 h after caffeine addition.
Isolation of mitotic chromatin
The polyamine buffer system used by Gasser and Laemmli (1987)
for purification of mitotic chromosomes was slightly modified and
utilized in this study to isolate mitotic chromatin from MUG cells.
The procedure was performed at 4°C. All solutions contained
0.25% thiodiglycol, 0.1 mM PMSF, 2 mg/ml aprotinin, 1 mg/ml
leupeptin and 1 mg/ml pepstatin. MUG cells were washed and
swollen at room temperature for 20 min in 0.5× buffer A (buffer
A is 15 mM Tris-HCl, pH 7.4, 0.2 mM spermine, 0.5 mM spermidine, 2 mM EDTA, pH 7.4, and 80 mM KCl). Swollen cells
were pelleted, resuspended in 0.5 ml of the same buffer and cooled
on ice. Ten ml of ice-cold lysis buffer (1× buffer A containing
0.1% digitonin) were added, and the lysate was homogenized on
ice in a Dounce homogenizer with a type A pestle.
The lysate was loaded onto a linear 35 ml glycerol gradient
(5% to 70% in buffer AS: 5 mM Tris-HCl, pH 7.4, 20 mM KCl,
20 mM EDTA, pH 7.4, 0.1 mM spermine, 0.25 mM spermidine,
0.1% digitonin) and spun in a swing out rotor at 200 g for 5 min
and at 750 g for 15 min. The gradient was collected from the top
and 2 ml fractions were assayed for the presence of chromatin.
Aliquots were spun onto microscope slides and analyzed by indirect immunofluorescence with CREST antibodies and staining
with Hoechst 33258 to visualize DNA. Samples of each fraction
were also analyzed for the pattern of CREST-antigens by
immunoblotting. Fractions containing small, well-separated chromatin particles, both centromeric and non-centromeric, were
diluted with buffer AS to reduce glycerol concentration to 10%
and spun at 3000 g for 20 min onto a 5 ml 70% glycerol cushion prepared with the same buffer. The supernatant was removed,
leaving 2 ml over the white material collected on the surface of
the glycerol cushion. Then, 20 ml of 89% Percoll in buffer AS
were added to the tube and the material was rehomogenized in a
Dounce homogenizer. The homogenate was subjected to isopycnic centrifugation in a Ti60 rotor at 40,000 r.p.m. for 30 min.
Chromatin formed a band in the lower part of the gradient, below
a flocculent mass of cytoplasmic debris, as determined by microscopic observation of 1.5 ml fractions.The chromatin-containing
fractions also contained most of the 80 kDa CREST antigen. They
were pooled and concentrated by spinning down onto a 70% glycerol cushion as described above.
Immunoaffinity purification of centromere
fragments
The buffer in which chromatin was suspended was changed to
PBS containing 1 mM MgCl 2 and 0.1% Triton X-100 (PBS/M/T).
This was done by spinning the chromatin down onto a 70% glycerol cushion through 15% glycerol solution in this buffer. Glycerol was removed by dialysis, the chromatin was homogenized
again and an equal volume of 2% BSA in PBS/M/T was added.
The suspension was centrifuged at 400 g for 7 min to remove
chromatin aggregates and larger pieces.
Centromeric DNA from kinetochore fragments
361
Immunobead reagent (Bio-Rad) linked to rabbit antibodies
specific to human immunoglobulins was used. The beads (0.3 ml
of suspension) were washed in PBS/M/T, blocked for 10 min in
the same solution containing 2% BSA (PBS/M/T/B), and pelleted
at 400 g for 5 min. Autoimmune serum SH diluted 1:400 in 2 ml
PBS/M/T/B was added to the pellet and the antibodies were
allowed to bind to the beads for 1 h. The beads were then washed
3 times for 10 min in the same buffer and added to the suspension of chromatin. After incubation for 2 h the beads with bound
chromatin were pelleted at 400 g for 5 min and washed with 10
ml of the same buffer 4 times for 5 min each, then once without
BSA. The pellet was resuspended in 0.2 ml TE buffer and used
for extraction of DNA.
ization procedure described by Lawrence et al. (1988) was used,
followed by one or two rounds of signal amplification with
biotinylated anti-avidin antibodies (Vector Laboratories) at 2
µg/ml and FITC-avidin DN (Vector Laboratories).
DNA cloning and analysis
RESULTS
All DNA manipulations were performed using conventional techniques (Sambrook et al., 1989). A 10 ng sample of isolated centromeric DNA was digested to completion with EcoRI and cloned
into Lambda Zap II vector (Stratagene). Three plaques were
picked at random and used for in vivo excision of pBluescript
plasmid containing the inserts. Fragments of the pCHC1 sequence
for use in in situ hybridization were generated by PCR, using
primer pairs corresponding to nucleotides 249-268 and 2306-2285;
3099-3118 and 4242-4222; 4544-5464 and 5798-5778.
DNA sequence analysis
DNA sequence analysis was performed using the EuGene/SAM
software package developed by the Molecular Biology Computational Resource at Baylor College of Medicine.
In situ hybridization
Probe DNA was labeled with biotin-11-dUTP by nick-translation
using the Photogene nucleic acid labeling kit (BRL). The hybrid-
Microscopy
Images were acquired by a SIT camera connected to a Zeiss Axiophot microscope with a Plan-Neofluar ×100 objective, preprocessed on an Argus-10 image processor (Hamamatsu Fotonics) and analyzed using Optimas software (Bioscan) on an IBM
PC.
Isolation of functional centromere fragments
Isolation of functional centromeric fragments involved
three steps: (1) in vivo chromosome fragmentation by
inducing the cells blocked at the G1/S-phase boundary of
the cell cycle to go into mitosis prematurely (Brinkley et
al., 1988); (2) isolation of total chromosome fragments; and
(3) immunoprecipitation of the centromere fragments using
antibodies of a CREST-scleroderma patient that are specific
to some antigens of the centromere/kinetochore region of
the chromosome (Earnshaw and Rothfield, 1985; Valdivia
and Brinkley, 1985).
It was reported before that if DNA replication is blocked
with hydroxyurea, cells can be induced to go into mitosis
prematurely by treatment with caffeine (Schlegel and
Pardee, 1986). In subsequent studies, Brinkley et al. (1988)
reported that under these conditions the centromere/kineto-
Fig. 1. Detached centromere-kinetochore complexes in MUG cells. (A) A MUG cell in anaphase, stained with Hoechst 33258. The
detached centromeric fragments (indicated by the asterisk) are segregating to the mitotic poles, while acentric chromatin (C) is displaced
to the cell periphery by the mitotic spindle. (B) Electron micrograph of a MUG cell in metaphase. Note the presence of trilaminar
kinetochore plates on the detached centromeric fragments (arrowheads).
362
I. I. Ouspenski and B. R. Brinkley
Fig. 2. Flow-chart of the procedure used for isolation of
centromere (CREST-positive) chromatin.
chore fragments detach from the rest of the chromosome.
These centromere fragments retain normal morphology and
remain functional, as evidenced by their ability to attach to
the mitotic spindle, align on the metaphase plate and
undergo apparently normal anaphase movements in vivo.
The total number of centromere fragments in treated cells
119
84
64
48
36
29
exceeds that of centromeres in intact G1 cells, indicating
that each fragment is only a portion of the centromere. Electron microscope observation shows that each detached fragment contains a distinct kinetochore plate and underlying
chromatin (Fig. 1). Our further observations indicate that
entire chromosome arms also undergo fragmentation under
these conditions (data not shown). We adapted the
polyamine chromosome isolation procedure of Gasser and
Laemmli (1987) for the isolation of dispersed mitotic chromatin. The strategy of the method is to homogenize hypotonically swollen cells in a buffer that stabilizes the compact structure of mitotic chromatin, and then to fractionate
the lysate by rate-zonal and isopycnic centrifugation (Fig.
2). Centrifugation through a glycerol gradient as the first
step allowed the separation of fine chromatin particles from
unlysed cells, nuclei, whole chromosomes and chromatin
aggregates (which sedimented to the lower part of the gradient), and soluble proteins (which remained in the supernatant). The chromatin was purified further by ‘banding’ in
an isopycnic gradient of Percoll, resulting in a preparation
that was essentially free of cytoskeletal debris, as determined by microscope observation (data not shown). However, some aggregation of chromatin occurred during this
step, necessitating a low-speed centrifugation to remove the
aggregates (Fig. 2).
Antiserum SH was selected for immunoaffinity purification because of the very low background produced when
used for immunofluorescent staining of the centromeres. In
immunoblotting experiments of CHO cell extracts, SH antiserum reacts with an 80-kDa antigen (which was determined to be centromeric by using antibodies affinity purified on the antigenic band on the blot; data not shown) and
two antigens in the 17-24 kDa range (Fig. 3A). It also shows
weak reaction with several soluble proteins that are
removed during the first centrifugation step. This might
explain our initial failure to immunopurify the centromere
fragments directly from crude cell lysates, without prior isolation of chromatin. Most of the 17-24 kDa antigens were
also solubilized under the lysis conditions used, leaving the
80-kDa centromeric protein, the only prominent antigen of
the chromatin detected by this autoantiserum. This resulted
Fig. 3. Distribution of CREST-antigens
in the course of centromeric chromatin
purification. (A) Immunoblot showing the
distribution of CREST-antigens after
fractionation of MUG cell homogenate
through a glycerol gradient. Fractions
were collected from the top and equal
volumes of material from each fraction
were subjected to SDS-electrophoresis
and immunoblotting with antiserum SH.
The direction of sedimentation is from
left to right. Relative molecular mass (in
kDa) of the marker proteins are indicated
at the left. (B) Dot-blot analysis of
CREST-antigens in the pellet (upper row)
and supernatant (lower row) obtained
after immunoprecipitation of mitotic
chromatin with CREST-antibody coated
beads. 1, 0.5 µg of total protein; 2, 2.5
µg; 3, 10 µg; 4 (supernatant only), 50 µg.
Centromeric DNA from kinetochore fragments
363
A
B
Fig. 4. Structure of the pCHC1 sequence. (A) Restriction map (P=PstI, H=HindIII), Alu repeats (solid bars) and fragments used as probes
in hybridization experiments. (B) Selected features of the sequence: A+T-rich regions (>80%); MAR consensus motif ATATTT(T)
(Cockerill and Garrard, 1986); A-box AATAAAYAAA (>9/10) (Gasser, 1986; Laemmli, 1986); T-box TTWTWTTWTT (>9/10) (Gasser
and Laemmli, 1986); topoisomerase II cutting site consensus sequences GTNWAYATTNATNNR (>13/15) (Sander and Hsieh, 1985)
in high specificity of the immunoprecipitation, leading to
recovery of virtually all of the immunopositive material
while leaving behind the rest of the chromatin (Fig. 3B).
Characterization of a centromere sequence,
pCHC1
The pellet obtained in the immunoprecipitation reaction was
used for extraction of DNA and subsequent cloning into a
lambda vector. Three phage plaques of the resulting library
were selected at random and assayed for genomic localization of cloned sequences by in situ hybridization to
metaphase chromosomes of the CHO cells. One of the
clones produced a weak signal at the centromere region,
while the other two gave a nearly uniform labeling of the
chromosomes, possibly due to the presence of interspersed
repetitive sequences. The first clone was designated pCHC1
and selected for further characterization.
This clone contains a 6 kb insert (Fig. 4) which has no
extensive homology to other known sequences, with the
exception of three copies of Alu-like repetitive elements.
The sequence apparently has no substantial coding capacity, as evidenced by the low magnitude of codon preference statistic (Gribskov et al., 1984) within open reading
frames (data not shown). Since our preparation of centromere enriched chromatin was obtained by virtue of its
affinity with CREST antibodies, and CENP-B was the
major CREST-antigen present in the preparation, we were
surprised to find no sequences homologous to the CENPB box consensus sequence as reported in human and mouse
DNA (Masumoto et al., 1989a; Wong and Rattner, 1988).
The closest matches to the YTTCGTTGGAARCGGGA
17mer (Masumoto et al., 1989a) was only 11 of 17
nucleotides. However, hybridization of two oligonucleotides that match the consensus (CTTCGTTG-
GAAACGGGA and TTTCGTTGGAAGCGGGA) to
genomic DNA from CHO cells also failed to reveal a positive signal (Fig. 5), suggesting that these variants of CENPB box do not exist in the Chinese hamster genome.
The centromere/kinetochore complex is an integral component of the scaffold (matrix) of metaphase chromosomes
(Earnshaw et al., 1984) and of interphase nuclei (Dacheng
He and B.R.B., unpublished). Although matrix attachment
Fig. 5. Hybridization of CENP-B box-like oligonucleotides to
hamster and human genomic DNA. (A) Hybridization with the
oligonucleotide CTTCGTTGGAAACGGGA. (B) Hybridization
with the oligonucleotide TTTCGTTGGAAGCGGGA (Masumoto
et al., 1989). Lane 1, lambda/HindIII; lane 2, DNA from human
cells (HeLa), 10 µg; lane 3, DNA from Chinese hamster cells
(CHO), 10 µg.
364
I. I. Ouspenski and B. R. Brinkley
Fig. 6. Genomic DNA of several mammalian species probed with
the hp5 fragment of pCHC1. DNA was digested with EcoRI and
PstI. Lane 1, pCHC1 plasmid, 100 pg; lane 2, kangaroo rat (Ptk1
cells), 20 µg; lane 3, African green monkey (COS-1 cells), 20 µg;
lane 4, Chinese hamster (CHO cells), 20 µg; lane 5, human (HeLa
cells), 20 µg; lane 6, mouse (L-M(TK−) cells), 20 µg; lane 7,
bovine (MDBK cells), 20 µg; lane 8, pCHC1 plasmid, 100 pg.
regions (MARs) share no obvious conserved sequence
elements, they exhibit some features that seem to
be conserved: they are A+T-rich, with clusters of
AATAAAYAAA (A-box) and TTWTWTTWTT (T-box)
motifs and topoisomerase II consensus cleavage sites
GTNWAYATTNATNNR (Gasser and Laemmli, 1986).
Analysis of the pCHC1 sequence reveals all of these features (Fig. 4).
When the whole 6 kb insert of pCHC1 was used as a
probe for Southern hybridization to genomic DNA from
CHO cells, a smear characteristic of highly repetitive
sequences was obtained (data not shown). Therefore, we
used subfragment hp5 of pCHC1 (Fig. 4) lacking the Alu
Fig. 7. In situ hybridization of pCHC1 fragments to metaphase
chromosomes of CHO cells. Chromosomes were stained with
Hoechst 33258 and pseudocolored red. Hybridization was
detected with FITC-conjugated avidin and pseudocolored yellow.
sequences for Southern hybridization with DNA of CHO
cells and cell lines from other species. The hybridization
pattern of hamster genomic DNA digested with EcoRI and
PstI was identical to that of the pCHC1 plasmid digested
with the same enzymes (Fig. 6). The presence of a higher
molecular weight band in both genomic and plasmid DNA
might be due to incomplete digestion. This sequence is
apparently not highly conserved, since it does not hybridize
to DNA of other mammalian species under medium stringency conditions (Fig. 6). The patterns of hybridization of
the hp6 fragment to DNA of CHO cells and of pCHC1
digested with EcoRI and PstI was also identical (data not
shown), suggesting that these sequences are not repetitive
in the Chinese hamster genome. Comparison of the intensity of hybridization of these two fragments to serial dilutions of CHO genomic DNA and pCHC1 plasmid DNA
indicates that these sequences are present on one or a few
(less than five) copies per haploid genome (data not shown).
The presence of Alu repetitive sequences in the pCHC1
clone probably explains the high background in our initial
attempts to localize this sequence by in situ hybridization.
In order to clarify the genomic location of the sequence,
we used a mixture of subfragments of pCHC1 that lacked
Alu sequences (Fig. 4) for fluorescence in situ hybridization
to CHO chromosomes. The hybridization pattern obtained
is a double dot at the centromere region of the longest chromosome of the complement (chromosome 1 - Ray and
Mohandas, 1976). As shown in Fig. 7, no other chromosome in the complement displays significant intensity of
hybridization.
DISCUSSION
Several procedures for isolation of fractions enriched in
centromere DNA from mitotic mammalian cells have been
developed. Wong and Rattner (1988) took advantage of the
reduced susceptibility of centromeric heterochromatin to
digestion with micrococcal nuclease to obtain a fraction of
mouse genomic DNA enriched in centromeric sequences.
This approach resulted in the cloning of several copies of
minor satellite DNA, one of the components of the mouse
centromere region. A similar approach was taken by
Masumoto et al. (1989b), who performed digestion of isolated mitotic chromosomes with a restriction endonuclease
under conditions of elevated salt concentration (0.3 M),
which resulted in preferential dispersion of chromosome
arms, while leaving the centromeric regions apparently
intact. This enabled these investigators to obtain a centromere-enriched fraction by sucrose gradient centrifugation. DNA cloned from this fraction contained sequences
of alphoid satellite, a prominent constituent of human centromeres (Masumoto et al., 1989b). All of these procedures
rely on the assumption that centromeric DNA sequences
remain largely intact and stay in association with the centromeric region or with CREST-antigens after nuclease
digestion. It has been well documented that the centromere
region, as opposed to chromosome arms, retains its morphology and apparently its protein composition when subjected to digestion with various nucleases (Rattner et al.,
1978; Lica and Hamkalo, 1983; Valdivia and Brinkley,
Centromeric DNA from kinetochore fragments
1985). It is less clear, however, whether all of the DNA
comprising this region is preserved under these conditions.
Thus, the sequence composition of DNA obtained from
such preparations may not be representative of the entire
centromere.
The method of chromosome fragmentation in vivo by
inducing premature chromatin condensation in cells
arrested with hydroxyurea overcomes this problem, as evidenced by the retention of mitotic function of the centromere fragments (Brinkley et al., 1988). The fact that one
of the first three clones picked at random from the library
obtained in this work hybridized to the centromere region
might be indicative of the high selectivity of this procedure.
Therefore, this approach has the potential of identifying
sequences on the basis of their association with functional
centromeres in vivo, and independently of any other
assumed properties of this region.
As inferred from the known centromere sequences of fission yeast (reviewed by Clarke, 1990), we might expect
DNA sequences in the centromere of mammalian chromosomes to be represented by repeats, as well as low copy
number sequences. Some of the centromere sequences can
be conserved, while others might be chromosome-specific
or species-specific. Some of these sequences should be
involved in the function of this segment, while others may
serve as ‘spacers’ to maintain the proper spatial organization of functional sequences. Some centromeric sequences
might be sequestered there, due to the reduced meiotic
recombination rate and lower evolutionary constraints in the
centromere. Therefore, it is not unexpected that the pCHC1
sequence fails to show any recognizable homology to other
known centromeric sequences of mammalian chromosomes, such as the alphoid satellite and classic satellites, or
mouse minor and major satellites. The only sequence
known to hybridize to the centromere region of most chromosomes in Chinese hamster cells is the ‘telomere-specific’
repeat (TTAGGG)n (Meyne et al., 1990; Zinkowski et al.,
1991). However, it does not hybridize to the larger metacentric chromosomes, where we detect the hybridization
signal of pCHC1. This suggests a different sequence organization of Chinese hamster chromosomes.
The absence of CENP-B box consensus sequences in the
Chinese hamster genome is surprising. In the human
genome, this sequence serves as a binding site for CENPB, an 80-kDa centromere-specific protein (Masumoto et al.,
1989a). This protein is probably involved in the mitotic
function of the centromere, since microinjection of antibodies leads to mitotic defects (Bernat et al., 1990; Simerly
et al., 1990). On the other hand, the results of expression
of truncated versions of this protein in mammalian cells
suggest that it might be redundant (Pluta et al., 1992).
CENP-B is highly conserved between humans and mouse
(Sullivan and Glass, 1991), and it seems likely that the
prominent 80 kDa CREST antigen that we detect in CHO
cells might be hamster CENP-B. At the same time, the
reported specificity of binding of the human protein to the
consensus 17mer sequence is very high, so that all studied
deviations abolish it (Masumoto et al., 1989a). It is also
unlikely that both CENP-B protein and the sequence it recognizes are different in hamster cells, since human chromosomes are known to be relatively stable in human-ham-
365
ster cell hybrids. To add to the controversy, the report by
Vig and Richards (1992) indicates that the distribution of
CENP-B protein on some chromosomes in a mouse cell line
might be different from that of the CENP-B box sequence,
even though it has been established that centromeric localization of CENP-B is determined by its binding to DNA
(Pluta et al., 1992; Yoda et al., 1992). The nature of these
apparently paradoxical relationships between the CENP-B
protein and CENP-B box sequences awaits further investigation.
There are three copies of monomeric hamster Alu repeats
in pCHC1. The hp5 and hp6 fragments of the clone that do
not contain Alu sequences are present in single copy (or
very few copies) in the Chinese hamster genome, as demonstrated by pattern and intensity of Southern hybridization.
In situ hybridization of a mixture of three fragments that
span a total of 4.4 kb of the 6 kb pCHC1 sequence, but do
not include the Alu repeats, gives a double dot signal at the
centromere region of only one pair of CHO chromosomes,
consistent with it being a single copy sequence. This does
not exclude the possibility that related, but not completely
conserved sequences might be present at the centromeres
of other chromosomes as well, since even alphoid centromeric DNA produces chromosome-specific signal in
human cells when used under conditions of high stringency.
The centromere-associated sequence pCHC1 apparently
is not expressed, as suggested by the low magnitude of
codon preference statistic for this region. This is consistent
with its location in the region of centromeric heterochromatin, which is believed to be transcriptionally silent. This
sequence contains several A+T-rich islands (30-75 bp, 8085% A+T content). This feature is characteristic of DNA
elements that mediate attachment of nuclear DNA to the
nuclear matrix or scaffold (Gasser and Laemmli, 1986;
Cockerill and Garrard, 1986). Although no single conserved
sequence has been identified in matrix associated regions
(MARs), they often contain multiple copies of topoisomerase II consensus recognition sequences, A and T-rich
motifs (A- and T-box, respectively) (Gasser and Laemmli,
1986) and a related motif identified by Cockerill and Garrard (1986). The pCHC1 sequence contains numerous
copies of these motifs (Fig. 4). MARs that have been studied so far are less than 1 kb in length and are separated by
loop domains spanning many kilobases (Phi-Van and
Stratling, 1990, and references therein). In pCHC1, however, the MAR-characteristic motifs are distributed throughout the central 3 kb of the fragment, forming several closely
located clusters. MAR sequence elements are often associated with transcription regulation elements (Gasser and
Laemmli, 1986) and are thought to delimit the ends of an
active chromatin domain (Phi-Van et al., 1990; LevyWilson and Fortier, 1989). Centromere regions of mammalian chromosomes are heterochromatic and transcriptionally silent, so attachment of DNA to nuclear matrix is
probably organized differently. Therefore, it is not clear at
present if the numerous MAR-like features found in pCHC1
are really indicative of an unusually long matrix-associated
region or some other unconventional chromatin feature.
Indirect support for this suggestion is provided by the fact
that prekinetochores (Brenner et al., 1981), as well as some
proteins of the centromere of metaphase chromosomes
366
I. I. Ouspenski and B. R. Brinkley
(Earnshaw et al., 1984) are integral components of the
nuclear matrix. Recent experiments in our laboratory
(Dacheng He and B.R.B., unpublished) indicate that a substantial portion of centromeric DNA is retained in association with the nuclear matrix. It is possible that the same
features of centromeric DNA are responsible for its resistance to nuclease digestion, both during preparation of the
nuclear matrix and in mitotic chromosomes. Characterization of other centromere sequences will undoubtedly help
clarify this and other questions of centromere structure.
The authors wish to thank Ray Zinkowski for helpful advice
regarding the generation of MUG cells, Dacheng He for helpful
discussions and advice on the work, Becky Scott for cell culture
work, help with immunofluorescence and image analysis and
advice on the manuscript, Alice Truong for participation in
sequencing, in situ and Southern hybridization experiments,
Hernan Grenett for performing hybridization with the CENP-B
box-like oligonucleotides, Andrey Zharkikh for help with the
analysis of the nucleotide sequence information, Donna Turner for
help with the electron microscopy, photography and preparation
of the illustrations, Mike Wise for help with preparation of the
manuscript and Craig Chinault and Greg May for comments on
the manuscript. This work was supported by a research grant from
the National Institutes of Health (CA41424). Sequence data available from GenBank, accession no. L12055.
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(Received 4 February 1993 - Accepted 22 March 1993)