2

The Mitotic Cell Cycle

P roliferating cells go through a regular cycle of events, the mitotic cell cy cle, in
wh ich the genetic material is duplicated and divided equally between two
daughter cells. This is brought about by the duplication of each chromosome to
form two closely adjacent sister chromatids, which separate from each other to
become two daughter chromosomes . These, along with the other chromosomes of
each set, are then packaged into two genetically identical daughter /luclei. The mo-
lecular mechan isms underlying the cell cycle are highly conserved in all organ-
isms with a nucleus (eukaryotes) . Many of the gene s and proteins involved in the
human cell cycle have been identified becau se of the ir high degree of nucleotide
and amino acid sequence similarity to homologous gene s and proteins in the
more easily studied budding yeast, Saccharomyces cerevisiae, in which the cell cycle
is more fully understood.

13
O. J. Miller et al., Human Chromosomes
© Springer-Verlag New York, Inc. 2001
'2 The Mitotic Cell Cycle

The Cell Cycle: Interphase,

Mitosis, and Cytokinesis
The interphase of the cell cycle (Fig. 2.1) is generally divided into three phases:
Ct (Cap I) , S (DNA synthetic), and G2 (Gap2). The mitotic part of the cycle
(M) is divided into five phases: prophase, prometaphase, metaphase, anaphase,
and telophase. Mitosis is followed by cytokinesis, the division of cytoplasm and
cell membrane required to complete the formation of two daughter cells . All
phases of the cell cycle are marked by an orderly progression of metabolic
processes. Cell differentiation is generally associated with a loss of proliferative
capacity (for example, in neurons or in muscle, kidney, or liver cells) ; these cells
are in a resting state, called Go. Some Go cells , such as lymphocytes or liver
cells , can be induced by a mitogen or growth factor to enter the cell cycle and
divide . Usually, extracellular mitogens bind to a membrane-bound receptor and
trigger the "immediate early response" genes, JUN, FOS, and MYC (Fig. 2.2).
These in turn trigger the sequential synthesis of cyclins and activation of spe-
cific cyclin-dependent kinases . These proteins (Fig. 2.1) and their genes (Table
2.1) regulate the cell cycle. They are also important in carcinogenesis, because
cell cycle regulatory genes can function as oncogenes (or cease functioning as

GO
cyclin D/CDK4 or 6

cyclin B/CDK1
G1

cyclin E/CDK2
cyclin AlCDK1 s a

cyclin AlCDK2

Figure '2.1 . The cell cycle involves sequential activation of cyclin -dependent
kinases (CDKs) by binding to cyclins . Cycl in D activates CDKs 4 and 6. The cycle
can be blocked at Gland G2 DNA damage checkpoints (a and b) or at a spindle
assembly checkpoint (c).

14
 The Cell Cycle: Interphase, Mitosis, and Cytokinesis

FOS
JUN

E A B

Go
------ R

Figure 2 .2. C hanges in gene expression levels during th e cell cycle . FOS, JUN, and
MYC are imme diate early respo nse genes whose expression is triggered by extracel -
lular stimuli. T he ensuing sequential expression of cyclins D 1, E, A, and B drives the
cell cycle (Roussel, 1998, with permission, Academic Press).

Table 2.1 . Major Cell Cycle Regulatory Proteins and the Genes That
Encode Them

Activato rs Inhibitors
Protein/gene Locat ion * Protein Gene Location
Cyclin A 4q26-q27 RBI RBi 13q13
Cyclin Bi Sql3-qter HDACI HDACi
Cyclin D1 l1q13 pS3 TP53 17p13
Cyclin D2 I2p13
Cyclin D3 6p21 C en eral Cyclin/CDK inhibitors
Cyclin E 19q12-q13 p21 CIPdWAFi 6p21 .2
CDK1 /CDC2 10q21 p27 KIPi 12p l2 -p 13
CDK2 12q1 3 pS7 KIP2 l1plS.St
CDK4 12q13
CDK6 7pl3--cen Sp ecific Cyclin/CDK4 and 6 inhi bitors
CDC2SA 3p21 pIS INK4B 9p21 -p22
p16, p19 INK4A 9p21-p22
p18 INK4 lp32

*Nomenclature of ISCN ( 199S)

t An imprinted gene (Chapter 21)
Source: Adapted from Hall and Peters ( 1997), with perm ission , Academic Press

15
:2 The Mitotic Cell Cycle

tumor suppressor genes) when activated (or inactivated) by specific transloca-

tions, deletions, or other types of genetic change (Chapters 27-28). Their aber-
rant function can also destabilize the genome and lead to multiple numerical and
structural chromosome changes (Chapter 26 ).

Cell Cycle Progression: Cy cl i n s

and Cyclin-Dependent Kinases
The cell cycle is driven by the sequential activation of key proteins by phospho-
rylation , the addition of phosphate groups to specific amino acid residues in the
proteins. Proteins that are called cyclins, because their synthesis is restricted to
particular parts of the cell cycle, play an important role (Figs. 2.1 and 2.2 ). They
activate a series of cyclin-dependent kinases (C D Ks), the enzymes that carry out the
phosphorylations. Cyclins D 1, D2 , and D3 activate CDK4 and CDK6 and drive
Go and G 1 cells through G 1. The cyclin ElCDK2 complex then drives the cells
through early 5, cyclin AlCDK2 through mrd-S. cycl in AlCDK 1 (also called
CDC2, cell divis ion cycle protein 2) through late S-early G2 , and cyclin
B/CDK 1 to the G2/M transition (Roussel, 1998 ). During cell differentiation, cell
lineages arise that are responsive to specific mitogenic signals, such as lym-
phokines in certain cla sses of lymphocytes and hormones in specific endocrine
tissues . Specific cell cy cle proteins are involved in this process. Thus, ovarian
follicle cells proliferate in response to follicle stimulating hormone , but this is
dependent upon the pre sence of a specific cyclin, D2 , wh ich activates the nec -
essary CDK4 or CDK6 (Sicinski et aI., 1996 ). M itogens of many types lead to
cell proliferation by binding to specifi c receptors and act ivating a cell cycle
signaling pathway (Fig. 2.3 ).
How does this cascade of phosphorylations lead to cell proliferation? Very
simply, the activated cyclin-dependent kinases add a few phosphate groups to
the retinoblastoma tumor suppressor protein, pRB (or RB). This reduces its
binding affinity for the E2F transcription factor, which is released in an active
form (Fig. 2.3 ). The underphosphorylated form of RB binds E2F, blocking its
function by a mechanism that was only recently discovered. Llnderphosphory-
lated RB in the RBlE2F complex binds a histone deacetylase (H D AC t), which
removes acetyl groups from the protruding tails of the adjacent nucleosomal hi s-
tones H3 and H4 (C hapter 5), creating a localized region of heterochromatin
(Fig. 2.4) . This reduces the access of transcription factors to the promoters of
the genes in this region and actively represses their transcription (M ag nagni-

16
 Cell Cycle Progression: Cyclins and Cyclm-Dependent Kinases

p16 1NK4a - ---I

E2F

~--r­
Responsive
Genes

Figure 2 .3. Signal transduction pathway from extracellular mitogen to the release
of active E2F transcription factor. This step can be blocked by the inhibitory protein
p16 , a product of the INK4A gene (Roussel, 1998, with permission , Academic Press).

jaulin et aI., 1998) . Other protein complexes with HDACI are involved in the
tissue-specific repression of genes (Chapter 5) , in X inactivation (Chapter 18),
and in imprinting (Chapter 21) .

?
•

E2F site

Figure 2.4. Model of repression of the transcription factor E2Fl in its E2F1IDPl
complex. RB binds to E2F and recruits HDAC 1 to E2F sites in promoters. This allows
HDAC 1 to deacetylate a target protein (X) locally, blocking transcription
(Magnagni-jaulin et aI., 1998, reprinted with permission from Nature, copyright
1998, Macmillan Magazines Limited) .

17
:2 The Mitotic Cell Cycle

When activated CDK4 and CDK6 phosphorylate RB, it releases E2F, which
then activates the synthesis of genes required for cell proliferation. One of these,
cyclin E, activates CDK2 kinase, leading to phosphorylation of still more pro -
teins and the initiation of the DNA synthetic (5) phase . DNA replication is so
important and has so many applications in cytogenetics that it is discussed more
fully in Chapter 3. Progression from 5 to G2 is marked by phosphorylation of
cyclin E, marking it for destruction by ubiquitin-mediated proteolysis. The same
fate is met by other short-lived regulatory proteins, including E2A, p53 , topoi-
somerase II, and the other cyclins in activated cyclin/CDK complexes, at the
appropriate times (Murray, 1995). This ensures that DNA is replicated only once
during each cell cycle .
Entry into mitosis (the G2/M transition) is mediated by the activation of a
cyclin-dependent kinase, the cyclin B/CDK 1 complex, which is also known as
maturation factor or mitosis promoting factor (MPF). MPF was discovered when
fusion of cells from different phases of the cell cycle revealed that cells in mitosis
contain a factor (MPF) that can drive interphase cells into mitosis , producing
premature chromosome condensation, or PCC (Fig. 23.3) . G 1 cells display long single
chromosome fibers, G2 cells (in which the DNA strands have replicated) have
long paired chromatids, and 5-phase cells have fragmented chromosomes. MPF
is activated by removal of phosphate groups by a phosphatase, CDC25, which
appears in late S and peaks in G2 . Microinjection of antibodies to CDC25 blocks
the cell's entry into mitosis (Lammer et al., 1998).
The activated kinase, cyclin B/CDK I, as MPF is now called, is a component of
mitotic chromatin that phosphorylates many proteins and thus triggers several
independent mitotic processes : breakdown of the nuclear envelope, chromosome
condensation, and spindle assembly. Phosphorylation of nuclear lamins leads to
disassembly of the nuclear envelope (Gerace and Blobel, 1980); phosphorylation
of histones HI and H3 leads to chromosome condensation; and phosphorylation
of stathmin, also called oncoprotein 18, leads to spindle assembly (Marklund
et al., 1996). At the metaphase-anaphase transition , an evolutionarily highly
conserved multi-protein complex called ubiquitin -protein ligase, or simply the
anaphase-promoting complex (APC) , catalyzes the conjugation of cyclin Band
related regulatory proteins to ubiquitin, targeting them for proteolysis (King et
al., 1995). In the absence of activated cyclin-dependent kinases to keep it phos -
phorylated, the RB protein loses its extra phosphate groups and again binds E2F,
inactivating it and completing the cycle . Table 2.1 lists key cell cycle regulatory
proteins and their gene loci. Many of these have been mapped by fluorescence in
situ hybridization (Chapter 8; Demetrick, 1995). Mutation, disruption , and dele -
tion of these genes playa major role in carcinogenesis (Chapters 26-28) .
18
 Cell Cycle Checkpoints

Cell Cycle Checkpoints

Genome integrity and the fidelity of mitosis are enhanced by a series of check-
points that monitor the successful completion of the successive phases of the cell
cycle and block further progression until each phase is completed (Fig. 2. I) . Cells
arrest in G I and G2 if their DNA has been damaged (by ionizing radiation, say),
and progression is delayed until DNA repair has occurred or celI death ensues.
DNA damage triggers the sequential activation of proteins (Fig. 2.5), first the
mutated in ataxia-telangiectasia (ATM) protein (Kastan et aI., 1992), then the p53
tumor suppressor protein, and then the GADD45 protein, which binds to the
proliferating cell nuclear antigen (PCNA) and blocks DNA replication (Chapter
3; Levine, 1997). There is also a spindle assembly checkpoint (Fig. 2. I) . CelIs
arrest in G2 and delay their exit from mitosis in response to spindle microtubule
disruption by spindle poisons such as colchicine, nocodazole, or benzimidazole.
A series of CDK inhibitors are involved in normal and genotoxic
damage-induced checkpoint regulation of the celI cycle (Table 2. I) . The
INK4A gene product, pl6, and two other proteins, pl5 and pl8, specifically
inhibit the cyclin D-dependent kinases CDK4 and CDK6 and can lead to G I
arrest. The KIPl gene product, p27, as welI as p2 I and the KIP2 gene product,
p57, inhibit all cyclin/CDK kinases and can arrest the cycle at various points.
The amount of the p27 protein is high in Go cells but is reduced by mitogens.
The tumor suppressor protein, p53 , stimulates production of p2 I and thus
blocks celI proliferation (Levine , 1997) . Mutation of the p53 gene is extremely

DNA damage

1
ATM (activated kinase)
~
~
p53 (activated)

1
.L
GADD45 (activated)
(inhibitory)

PCNA - DNA replication

Figure 2 .5. A signaling pathway by which DNA damage activates a cell cycle
checkpoint and blocks DNA replication .

19
:2 The Mitotic Cell Cycle

important in carcinogenesis because of its key role both in DNA damage

checkpoints and at spindle assembly checkpoints. These checkpoints are dis-
cussed more fully in Chapter 26 because of the critical role they play in genome
stability.

Prophase
In interphase nuclei , chromosomes are invisible except for occasional clumps of
chromatin, called chromocenters, which vary in size and shape. One or more
nucleoli are usually visible . At prophase, the chromosomes become visible as
long thin threads that gradually shorten and thicken (co nde nse) as mitosis pro-
gresses (Fig. 2.6 ). Chromosome condensat ion at the onset of prophase requires
topoisomerase II (T a pa II), as sho wn by immunodepletion and complementa-
tion studies. Tapa II (scaffold protein 1, or SCI ) is the most abundant com-
ponent of the chromosome scaffo ld (C hapter 5). It reaches its peak level and
activity at the G2 /M transition. It binds to DNA at AT-rich sites called scaffold
attachment regions (SARs) and appears to aggregate these, aiding in chromosome
condensation (Adach i et al., 199 I) . As its name implies, TOPO II also intro-
duces transient double-strand nicks that permit separatio n of the intertwined
newly replicated DNA of sister chromatids. The seco nd mo st abundant chro-
mosome scaffold prote in, SCII , is also invo lved in chromosome condensation .
It belongs to the h ighly conserved SMC (stab ility of minichromosomes)
family of proteins found in all eukaryotes. Their structure resembles that of
mechanochemical (mo to r) proteins like myosin and kinesin , suggesting that
chromosome condensation involves a motor function to bring chromosome
regions more tightly together (Kos h land and Strunnikov, 1996).
During mitosis, RNA synthesis is completely shut down, as first shown autora-
diographically in the 1960s by the absence of incorporation of radioactively
labeled [3H]uridine, which is a specific precursor of RNA. The mechanism of
shutdown involves displacement of all the sequence-specific transcription factors
from the promoter seque nces of the genes, perhaps due in part to the action of
the SMC protein complex, which can bring about renaturation of any single
stranded regions in DNA (Sutani and Yanaguta, 1997). The SMC complex is
also associated with the deacetylation and phosphorylation of the core histones
that accompany chromosome condensation (Chapter 5). Nucleoli diminish in
size as a result of the near cessation of ribosomal RNA synthesis, which is absent
from prometaphase to late anaphase (C hapter 4).

20
 Prophase

INTERPHASE
Chromosome Reproduction
f Doubling of the DNA Content

TERPHASE - - - - - - - PROPHASE - - - ----

PROM ETAPHAS E lETAPHASE - --- A APHASE

TELOPHASE II TE RPHASE

- -- A API-lASE - - TELOPHASE

Figure '2.6. Mitotic cycle in cultured lymphocytes (Feulgen-stained squash preparations).

21
:2 The Mitotic Cell Cycle

Prometaphase, Centrosomes,
and the Mitotic Spindle
Prometaphase is associated with the breakdown of the nuclear membrane and the
disappearance of nucleoli. The chromosomes continue their condensation, and
a new structure, the mitotic spindle, is organized between the two centrosomes.
The centrosome is the major microtubule organizing center (MTOC) in both inter-
phase and mitotic cells. It consists of the highly structured centriole and its sur-
rounding proteinaceous matrix, and contains specific proteins such as y-tubulin
and centrosomins A and B (Petzelt et aI., 1997). The y-tubulin anchors the minus
(slow-growing) end of microtubules to the pericentriolar material of the centro-
some . The plus (fast-growing) end of each microtubule projects into the cyto-
plasm during interphase and into the spindle during mitosis (Zheng et al., 1995).
The centrosome duplicates itself once per cell cycle, starting at the G t/S tran-
sition . Cyclin E/CDK2 is essential for centrosome duplication in S, ensuring
proper coordination of spindle assembly with other events of the cell cycle (Fig.
2.1 i Hinchcliffe et al., 1999). At the G2/M tran sition (prophase), the duplicated
centrosomes separate and go to opposite poles of the nucleus . A kinesin -related
motor protein, Eg5, is essential for this process, as microinjection of antibodies
to Eg5 arrests cells in mitosis with a monopolar spindle. To act as a motor
protein, Eg5 must first be phosphorylated by the cyclin-dependent kinase
CDK 1i this coordinates its activity with other events at the G2/M transition
(Blangy et al., 1995). The separated centrosomes reorganize the microtubules of
the cell by the coordinated dissolution of all the cytoplasmic microtubular appa -
ratus and assembly of the mitotic spindle, with one centrosome at each pole. A
complex of the nuclear mitotic apparatus protein (NuMA) and the cytoplasmic
motor protein , dynein, is essential for assembly of the spindle. NuMA tethers
bundles of microtubules and anchors them to the spindle poles . At telophase,
NuMA is released from each pole as part of the dissolution of the spindle
(Merdes et aI., 1996).

Metaphase, Anaphase, and Telophase

The chromosomes reach their maximal state of compaction during metaphase.
The spindle apparatus mediates the orchestrated movement of chromosomes
during mitosis. Microtubules emanating from the spindle pole are captured by a

22
 Metaphase, Anaphase, and Telophase

special structure, the kinetochore (Chapter 4), which forms at the centromere of
each chromatid and attaches it to the mitotic spindle. The kinetochore protein
CENP-E belongs to the kinesin family of motor proteins. After phosphorylation
by activated CDK 1, it participates in the chromosome movements at metaphase
and early anaphase (Liao et ai, 1994). CENP-E and other motor proteins of the
spindle fibers pull the kinetochores to a plane, called the metaphase plate, that is
midway between the two poles of the spindle. The chromosomes reach their
maximum condensation, to ,OOO-fold shorter than the DNA molecule itself, as
anaphase begins .
The onset of anaphase is controlled by a specific mitotic spindle assembly check-
point (Chapter 26) that monitors kinetochore attachment to the spindle and is
mediated by an inhibitory signal produced by kinetochores that have not yet
captured the necessary spindle microtubules (Wells, 1996). Apparently, it is the
tension produced at the kinetochore by the bipolar attachment of spindle fibers
that is monitored (Nicklas, 1997). The proteins required for proper checkpoint
function and the genes that encode them are highly conserved during evolution.
Mutations that enable cells to proceed through mitosis despite depolymeriza-
tion of the spindle by nocodazole or benzimidazole have recently defined three
MAD (mitosis arrest deficient) and three BUB (budding uninhibited by benz-
imidazole) spindle assembly checkpoint genes in budding yeast, and several of
their human homologs have already been identified. One of them , MAD2, forms
a complex with APC and the mitotic regulator CDC20 to block the activation
of APC (Fang et al., 1998). Another, BUB 1, may be the tension-sensitive kinase
that provides the signal for anaphase to begin .
Once the spindle is assembled and attachment of each chromosome to spindle
fibers from both poles is completed, producing tension, the transition from
metaphase to anaphase occurs . The process is still very poorly understood. Two
cell division cycle (C DC ) proteins that co-localize to the spindle, CDC16 and
CDC27, are required. When antibodies to human CDC27 are injected into
HeLa (human cervical cancer) cells, the transition from metaphase to anaphase
is blocked (Tugendreich et aI., 1995).
During anaphase, the kinetochores of sister chromatids separate and the
movement of motor proteins along the shortening spindle microtubules drags
the daughter chromosomes towards opposite poles of the elongating spindle
(Fig. 2.6). One motor protein, MKLP- 1 (mitotic kinesin-like protein- 1), associ-
ates specifically with the mitotic spindle. It bundles anti parallel microtubules and
moves them apart at about the same speed as spindle elongation, suggesting that
it provides the force for anaphase B, spindle elongation (Nislow et aI., 1992).

23
2 The Mitotic Cell Cycle

The separation of sister chromatids requires ubiquitin-mediated breakdown of

proteins by APC. It also requires topoisomerase II (Tapa II), to untangle the
tangled, or catenated, strands of newly replicated DNA, and the Tapa 11-
interacting protein, BRRN1, the human homologue of the barren protein in
Drosophila (Bhat et aI., 1996). Inhibitors of Tapa II block sister chromatid
separation; partial inhibition can lead to nondisjunction (Downes et aI., 199 I).
The regulation of anaphase is still rather poorly understood, but study of
further mutations should be helpful. Two families have been described in which
sister chromatids frequently separated prematurely in two unrelated infants,
leading to nondisjunction, multiple mosaic trisomies and monosomies, and
severe malformations. All four parents had a milder form of premature chromatid
separation and were presumably heterozygous for the mutation that was
homozygous in their severely affected offspring (Kajii et aI., 1998).
During telophase, the mitosis-to-interphase transition, the chromosomes
decondense, the mitotic spindle dissipates , the microtubules reorganize into a
cytoplasmic microtubule network; the nuclear envelope reassembles, and nucle-
oli reappear. Nucleoli tend to fuse, but the largest number observed at telophase
indicates the number and chromosomal location of active ribosomal RNA gene
clusters , the nucleolus organizers (Chapter 4) . Telophase is usually followed by cyto-
plasmic division (cytokinesis), after which the cells are once more in interphase.
A novel human protein , PRe 1, associates with the mitotic spindle and localizes
to the cell midbody during cytokinesis. Microinjection of anti-PRCI antibodies
into human HeLa cells does not affect nuclear division (mitosis) but blocks
cytokinesis and cell cleavage (liang et aI., 1998).

Nondisjunction, Loss of
Chromosomes, and Mosaicism
The orderly segregation of daughter chromosomes in anaphase may fail for
various reasons, such as mutation of one of the many genes required for mitosis
or advancing maternal age, which has a profound effect of unknown cause. The
inclusion of both daughter chromosomes in the same nucleus , by whatever
mechanism, is called nondisjunction . In this process , one daughter cel1 receives an
extra copy of a chromosome, becoming trisomic for this chromosome, and the
other daughter cel1 loses a copy, becoming monosomic for this chromosome. One
or both daughter chromosomes may lag behind at anaphase and reach neither
pole . Such laggards form micronuclei in telophase. Micronuclei usual1y do not

24
 References

enter mitosis, and consequently any chromosomes included in a micronucleus

are lost from the complement.
If the trisomic and monosomic cells that arise in somatic tissues through
mitotic nondisjunction or chromosome loss are viable, the result is chromosomal
mosaicism, the presence in one individual of clones of cells with differing chro-
mosome constitutions. Mosaicism may be found in a single tissue , multiple
tissues , or the entire organism, depending on the time in development at which
nondisjunction occurred. Given the frequencies of nondisjunction and chromo-
some loss throughout life, we are all mosaics to some degree .

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