Download Cell Division - HCC Learning Web

CAMPBELL
BIOLOGY
TENTH
EDITION
Reece • Urry • Cain • Wasserman • Minorsky • Jackson
12
The Cell Cycle
Lecture Presentation by
Nicole Tunbridge and
Kathleen Fitzpatrick
© 2014 Pearson Education, Inc.
Figure 12.1
How do dividing cells distribute chromosomes to daughter cells?
The ability of organisms to reproduce distinguishes living
things from nonliving.
The continuity of life is based on the reproduction of cells, or
cell division.
Chromosomes (blue) are
moved by cell machinery
(red) during division of a rat
kangaroo cell.
Figure12.2
Cell Division
• In unicellular organisms, division of one cell reproduces the entire
organism
• In multicellular organisms depend on cell division for:
– Development from a fertilized cell
– Growth
– Repair
• Cell division is an integral part of the cell cycle, the life of a cell from
its origin in the division of a parent cell until its own division into two
© 2014 Pearson Education, Inc.
Cell division results in genetically identical
daughter cells
• Cell division requires the distribution of identical genetic
material-DNA to two daughter cells.
– The special type of cell division meiosis that produces
sperm and eggs results in daughter cells that are not
genetically identical.
• DNA is passed along, without dilution, from one generation
to the next.
• All the DNA contain cell’s genetic information in a cell
constitutes called as genome. DNA molecules in a cell are
packaged into chromosomes.
-In prokaryotes, the genome is often a single circular DNA
molecule.
-In eukaryotes, the genome consists of several long DNA
molecules.
© 2014 Pearson Education, Inc.
Cell Division
Figure 12.3
• A dividing cell duplicates its DNA, allocates the
two copies to opposite ends of the cell, and only
then splits into daughter cells.
• A human cell must duplicate about 2 m of DNAa length about 250,000 times grater than the
cell’s diameter and separate the two copies so
that each daughter cell ends up with a complete
genome.
© 2014 Pearson
Education, Inc.
Cellular Organization of the Genetic
Material
• DNA molecules in a cell are packaged into
chromosomes
• Every eukaryotic species has a characteristic
number of chromosomes in each cell nucleus.
• Human somatic cells (all body cells except sperm
and egg) have 46 chromosomes, made up of two
sets of 23 (one from each parent).
• Reproductive cells or gametes (sperm or eggs)
have one set of 23 chromosomes, half the number
in a somatic cell.
• Eukaryotic chromosomes are made of chromatin,
a complex of DNA and associated protein.
© 2014 Pearson Education, Inc.
Distribution of Chromosomes During
Eukaryotic Cell Division
• When a cell is not dividing, each chromosome is
in the form of a long, thin chromatin fiber.
• Before cell division, the chromatin condenses,
coiling and folding to make a smaller package.
• Each duplicated chromosome consists of two
sister chromatids, which contain identical
copies of the chromosome’s DNA.
© 2014 Pearson Education, Inc.
• The centromere is the narrow
“waist” of the duplicated
chromosome, where the two
chromatids are most closely
attached.
• The part of a chromatid on
either side of the centromere is
called an arm of the chromatid.
• Later in cell division, the sister
chromatids separate and move
into two new nuclei at opposite
ends of the parent cell.
– After the sister chromatids
separate, they are
considered individual
chromosomes.
– Each new nucleus receives
a group of chromosomes
identical to the original
group in the parent cell.
Figure12.4 & 12.5
© 2014 Pearson Education, Inc.
Eukaryotic Cell Division
• Eukaryotic cell division consists of:
1. Mitosis- the division of the nucleus
2. Cytokinesis- the division of the cytoplasm
– These processes start with one cell and produce two cells that are
genetically identical to the original parent cell.
– Each of us inherited 23 chromosomes from each parent: one set in
an egg and one set in sperm, for a total of 46.
– The chromosomes combine in the nucleus of a single cell when a
sperm unites with an egg to form a fertilized egg, or zygote.
– The zygote undergoes cycles of mitosis and cytokinesis to produce
a fully developed multicellular human made up of 200 trillion
somatic cells.
– Mitosis continues every day to replace dead and damaged cells.
– Essentially, mitosis produces clones—cells with identical genetic
information, mitosis produce 2cells and maintain chromosome # 46
in each cell after division,.
© 2014 Pearson Education, Inc.
Meiosis
• In contrast, gametes (eggs or sperm) are
produced only in gonads (ovaries or testes) by a
variation of cell division called meiosis.
• Meiosis yields four non-identical daughter cells,
each with half the chromosomes of the parent.
• In humans, meiosis reduces the number of
chromosomes from 46 to 23.
• Fertilization fuses two gametes together and
returns the number of chromosomes to 46
again.
© 2014 Pearson Education, Inc.
The mitotic phase alternates with
interphase in the cell cycle
•
The mitotic (M) phase of the cell cycle,
which includes mitosis and cytokinesis,
alternates with the much longer
interphase.
– Interphase accounts for about 90% of the
cell cycle.
•
Interphase has three subphases:
– the G1 phase “first gap”
– the S phase “synthesis”-chromosomes
are duplicated
– the G2 phase “second gap”
– The daughter cells may then repeat the
cycle.
– The cell grows during all three phases,
but chromosomes are duplicated only
during the S phase
•
•
During all three sub phases, the cell
grows by producing proteins and
cytoplasmic organelles such as
mitochondria and endoplasmic reticulum.
A typical human cell might divide once
every 24 hours.
© 2014 Pearson Education, Inc.
Figure 12.6
Mitosis
• Mitosis is divided into five phases:
– Prophase
– Prometaphase
– Metaphase
– Anaphase
– Telophase
• Cytokinesis begins during telephase and
completes mitosis.
• In 1882, the German anatomist Walther Flemming
developed dyes to observe chromosomes during mitosis
and cytokinesis
© 2014 Pearson Education, Inc.
Figure 12.7a
10 μm
In Animal Cell-Mitosis-preview
G2 of Interphase
Centrosomes
(with centriole
pairs)
Nucleolus
Chromosomes
(duplicated,
uncondensed)
Nuclear
envelope
Plasma
membrane
Prophase
Early mitotic
spindle
Aster
Centromere
Two sister chromatids
of one chromosome
Prometaphase
Fragments
of nuclear
envelope
Kinetochore
Nonkinetochore
microtubules
Kinetochore
microtubule
10 μm
Figure 12.7b
Metaphase
Anaphase
Metaphase
plate
Cleavage
furrow
Daughter
chromosomes
Spindle
Telophase and Cytokinesis
Centrosome at
one spindle pole
Nuclear
envelope
forming
Nucleolus
forming
Figure 12.11
In Plant Cell-Mitosis-preview
Plant Cell
Nucleus
Chromatin
condensing
Nucleolus
1 Prophase
Chromosomes
2 Prometaphase 3 Metaphase
Cell plate
4 Anaphase
10 µm
5 Telophase
Microtubules are responsible for the
separation of chromosomes during cell
division
Figure 6.22
• Microtubules grow out from
a centrosome near the
nucleus.
• The centrosome is a
“microtubule-organizing
center”.
• In animal cells, the
centrosome has a pair of
centrioles, each with nine
triplets of microtubules
arranged in a ring.
• Before an animal cell
divides, the centrioles
replicate.
© 2014 Pearson Education, Inc.
Late Interphase
• In interphase, the
chromosomes have
been duplicated but
are not condensed.
• A nuclear membrane
bounds the nucleus,
which contains one or
more nucleoli.
• The centrosome has
replicated to form two
centrosomes.
• In animal cells, each
centrosome is made
up of two centrioles.
© 2014 Pearson Education, Inc.
Prophase
• In prophase, the
chromosomes are tightly
coiled, with sister chromatids
joined together.
• The nucleoli disappear.
• The mitotic spindle begins to
form. It is composed of
centrosomes and the
microtubules that extend from
them.
• The radial arrays of shorter
microtubules that extend from
the centrosomes are called
asters.
• The centrosomes move away
from each other by
lengthening microtubules.
© 2014 Pearson Education, Inc.
Prometaphase
• During prometaphase, the
nuclear envelope fragments,
and microtubules from the
spindle interact with the
condensed chromosomes.
• Each of the two chromatids of
a chromosome has a
kinetochore, a specialized
protein structure located at the
centromere.
• Kinetochore microtubules from
each pole attach to one of two
kinetochores.
• Nonkinetochore microtubules
interact with those from
opposite ends of the spindle.
© 2014 Pearson Education, Inc.
Metaphase
• The centrosomes are now
at opposite poles of the cell.
• The spindle fibers push the
sister chromatids until they
are all arranged at the
metaphase plate, an
imaginary plane equidistant
from the poles, defining
metaphase.
• For each chromosome the
kinetochores of the sister
chromatids are attached to
kinetochore microtubules
coming from opposite poles.
© 2014 Pearson Education, Inc.
Anaphase
• The sister chromatids
separate, becoming
daughter chromosomes.
• The separated
chromatids move to
opposite ends of the cell
as their kinetochore
microtubules shorten.
• By the end of anaphase
the two poles of the cell
have identical
chromosomes.
© 2014 Pearson Education, Inc.
Telophase
• Two daughter nuclei
form.
• The nuclear envelope
forms .
• The chromosomes
become less
condensed.
• Mitosis-the division of
one nucleus into two
identical nuclei, is
complete.
© 2014 Pearson Education, Inc.
Cytokinesis
• The division of the
cytoplasm begins
in telophase.
• A cleavage furrow
forms that
“pinches” the cell in
two.
© 2014 Pearson Education, Inc.
The Mitotic Spindle: A Closer Look
• The mitotic spindle is a structure made of microtubules
that controls chromosome movement during mitosis
• In animal cells, assembly of spindle microtubules begins
in the centrosome, the microtubule organizing center
• The centrosome replicates during interphase, forming
two centrosomes that migrate to opposite ends of the
cell during prophase and prometaphase
• An aster (a radial array of short microtubules) extends
from each centrosome
• The spindle includes the centrosomes, the spindle
microtubules, and the asters
• During prometaphase, some spindle microtubules attach
to the kinetochores of chromosomes and begin to move
the chromosomes
Figure 12.8
Sister
chromatids
Aster
Centrosome
Metaphase
plate
(imaginary)
Kinetochores
Microtubules
Overlapping
nonkinetochore
microtubules
Chromosomes
Kinetochore
microtubules
Centrosome
1 µm
0.5 µm
Kinetochores
• Each sister chromatid has a kinetochore of proteins and
chromosomal DNA at the centromere.
• The kinetochores of the joined sister chromatids face in
opposite directions.
• When a chromosome’s kinetochore is “captured” by
microtubules, the chromosome moves toward the pole from
which those microtubules come.
• At metaphase, the chromosomes are all lined up at the
metaphase plate, a plane midway between the spindle’s two
poles
© 2014 Pearson Education, Inc.
What is the function of the nonkinetochore
microtubules?
• Nonkinetochore microtubules are responsible for lengthening
the cell along the axis defined by the poles.
• Nonkinetochore microtubules interdigitate and overlap across
the metaphase plate.
• During anaphase, the area of overlap is reduced as motor
proteins attached to the microtubules walk them away from
one another, using energy from ATP.
• As the microtubules push apart, they lengthen by the addition
of new tubulin monomers to their overlapping ends, allowing
continued overlap.
• In anaphase the cohesins are cleaved by an enzyme called
separase
• Sister chromatids separate and move along the kinetochore
microtubules toward opposite ends of the cell
• The microtubules shorten by depolymerizing at their
kinetochore ends
© 2014 Pearson Education, Inc.
Figure 12.9
EXPERIMENT
Kinetochore
Spindle
pole
Mark
RESULTS
CONCLUSION
Microtubule
Chromosome
movement
Motor protein
Chromosome
Kinetochore
Tubulin
subunits
Cytokinesis: A Closer Look
• In animal cells cytokinesis occurs (following
mitosis) by a process known as cleavageforming a cleavage furrow
• In plant cells, a cell plate forms (from Golgi
vessicles) during cytokinesis
© 2014 Pearson Education, Inc.
Cytokinesis in Animal Cells
• On the cytoplasmic
side of the cleavage
furrow is a contractile
ring of actin
microfilaments
associated with
myosin.
• Contraction of the ring
pinches the cell in
two.
Figure 12.10
© 2014 Pearson Education, Inc.
Cytokinesis in Plant Cells
• During telophase, vesicles
from the Golgi apparatus
move along microtubules
to the middle of the cell,
where they coalesce to form
a cell plate.
• Cell wall materials carried in
the vesicles collect in the cell
plate as it grows.
• The plate enlarges until its
membranes fuse with the
plasma membrane at the
perimeter.
• The contents of the vesicles
form new cell wall material
between the daughter cells.
© 2014 Pearson Education, Inc.
Binary Fission-Bacterial cell
• Asexual reproduction of single-celled eukaryotes, such
as an amoeba, includes mitosis and occurs by a type of
cell division called binary fission- “division in half.”
• Most bacterial genes are located on a single bacterial
chromosome that consists of a circular DNA molecule
and associated proteins, they still have large amounts of
DNA that must be copied and distributed equally to two
daughter cells.
• Prokaryotes (bacteria and archaea) reproduce by a type
of cell division called binary fission
• In binary fission, the chromosome replicates (beginning
at the origin of replication), and the two daughter
chromosomes actively move apart
• The plasma membrane pinches inward, dividing the cell
into two
© 2014 Pearson Education, Inc.
Bacterial Cell Division by Binary Fission
• In the bacterium
Escherichia coli, the
process of cell division
begins when the DNA of
the bacterial chromosome
starts to replicate at a
specific place on the
circular chromosome- the
origin of replication site,
producing two origins.
© 2014 Pearson Education, Inc.
Figure 12.12
Bacterial Cell Division by Binary Fission
• As the chromosome
continues to replicate,
one origin moves
toward each end of
the cell.
• While the
chromosome is
replicating, the cell
elongates.
© 2014 Pearson Education, Inc.
Bacterial Cell Division by Binary Fission
•
•
•
When replication is complete, the
plasma membrane grows pinches
inward to divide the parent E. coli cell
into two daughter cells, each with a
complete genome.
Researchers use fluorescence
microscopy to observe the movement
of bacterial chromosomes.
– The movement is similar to the
2
poleward movements of the
centromere regions of eukaryotic
chromosomes.
– However, bacterial
chromosomes lack visible
mitotic spindles or even
microtubules.
In most bacterial species studied, the
two origins of replication end up at
opposite ends of the cell or in some
other very specific location, possibly
anchored there by one or more
proteins.
© 2014 Pearson Education, Inc.
1
Chromosome
replication
begins.
2
Replication
continues
3 Replication
finishes.
4
Two daughter
cells result.
Mitosis in eukaryotes may have evolved
from binary fission in bacteria
•
•
•
•
There is evidence that mitosis had its
origins in bacterial binary fission.
The fact that some of the proteins
involved in bacterial binary fission are
related to eukaryotic proteins that
function in mitosis supports this
hypothesis.
As eukaryotes evolved, binary fission
probably gave rise to mitosis.
Possible intermediate evolutionary
steps are seen in the division of two
types of unicellular algae.
– In dinoflagellates, replicated
chromosomes are attached to the
nuclear envelope and separate as the
nucleus elongates prior to cell division.
– In diatoms, a spindle within the nucleus
separates the chromosomes.
•
In most eukaryotic cells, the nuclear
envelope breaks down and a spindle
separates the chromosomes.
© 2014 Pearson Education, Inc.
Fig. 12.13
The eukaryotic cell cycle is regulated by a
molecular control system
• The timing and rates of cell division in different
parts of an animal or plant are crucial for normal
growth, development, and maintenance.
• The frequency of cell division varies with cell type.
– Some human cells divide frequently throughout life
(skin cells).
– Others human cells have the ability to divide but
keep it in reserve (liver cells).
– Mature nerve and muscle cells do not appear to
divide at all after maturity.
– Cancer cells manage to escape the usual controls
on the cell cycle
© 20114 Pearson Education, Inc.
Evidence for Cytoplasmic Signals
• The cell cycle appears to be driven by
specific chemical signals present in the
cytoplasm
• Some evidence for this hypothesis comes
from experiments in which cultured
mammalian cells at different phases of the
cell cycle were fused to form a single cell
with two nuclei
© 2014 Pearson Education, Inc.
Figure 12.14
Experiment
Experiment 1
S
G1
S
S
Experiment 2
M
G1
Results
When a cell in the S
phase was fused
with a cell in G1,
G1 nucleus
immediately entered
S phase and DNA
was synthesized.
Conclusion
M
M
When a cell in the
M phase was fused with
a cell in G1, G1 nucleus began
mitosis without
chromosome
duplication.
Molecules present in the cytoplasm control
the progression to S and M phases.
Cytoplasmic signals drive the cell cycle
• The sequential events
of the cell cycle are
directed by a distinct
cell cycle control
system
– Cyclically operating
molecules trigger and
coordinate key events in
the cell cycle.
– The control cycle has a
built-in clock, but it is
also regulated by
external adjustments
and internal controls.
• The cell cycle has
specific checkpoints
where the cell cycle
stops until a go-ahead
signal is received
© 2014 Pearson Education, Inc.
Figure 12.15
The Cell Cycle Clock: Cyclins and
Cyclin-Dependent Kinases
• The cell cycle regulatory molecules are mainly proteins of two
types: protein kinases and cyclins.
– Protein kinases are enzymes that activate or inactivate other
proteins by phosphorylating them.
– Particular protein kinases give the go-ahead signals at the G1 and
G2 checkpoints.
• The kinases that drive the cell cycle are present at constant
concentrations but require the attachment of a second protein, a
cyclin, to become activated.
– Levels of cyclin proteins fluctuate cyclically.
– Because of the requirement for binding of a cyclin, the kinases
are called cyclin-dependent kinases, or Cdks.
• Cyclin levels rise sharply throughout interphase and then fall
abruptly during mitosis.
© 204 Pearson Education, Inc.
Figure 12.16
Peaks in the activity of the cyclin-Cdk
complex, maturation-promoting factor,
(MPF) correspond to peaks in cyclin
concentration
M
G1
S
G2
M
G1
S
G2
M
G1
Cdk
MPF
activity
Cyclin
concentration
Degraded
cyclin
Cdk
Cyclin is
degraded
MPF
Time
(a) Fluctuation of MPF activity and cyclin
concentration during the cell cycle
Cyclin
G2
checkpoint
(b) Molecular mechanisms that help regulate
the cell cycle
Peaks in the activity of MPF correspond to peaks
in cyclin concentration
• MPF (“maturation-promoting factor” or “M-phasepromoting factor”) triggers the cell’s passage past
the G2 checkpoint to the M phase.
– MPF promotes mitosis by phosphorylating a variety of
other protein kinases.
– MPF acts both directly as a kinase and indirectly by
activating other kinases.
– MPF stimulates fragmentation of the nuclear
envelope by phosphorylation of various proteins of
the nuclear lamina during prometaphase of mitosis.
– MPF also contributes to the molecular events
required for chromosome condensation and spindle
formation during prophase.
– MPF triggers the breakdown of cyclin, reducing cyclin
and MPF levels during mitosis and inactivating MPF.
© 2014 Pearson Education, Inc.
Molecular control of the cell cycle
1
2
3
4
5
Cyclin synthesis occurs in
late S phase through G2.
Cyclin molecules combine
with Cdk to make MPF.
The cell passes from the
G2 checkpoint into mitosis.
MPF promotes mitosis by
P proteins.
Cyclin is degraded during
anaphase, terminating M
phase. The cell enters
G 1.
Cdk is recycled during G1.
5
1
4
2
3
Figure 12.17
© 2014 Pearson Education, Inc.
Stop and Go Signs: Internal and External
Signals at the Checkpoints
• Many signals registered at checkpoints come from
cellular surveillance mechanisms within the cell
• Checkpoints also register signals from outside
the cell
• Three important checkpoints are those in G1, G2, and M
phases
• For many cells, the G1 checkpoint seems to be the most
important
• If a cell receives a go-ahead signal at the G1 checkpoint,
it will usually complete the S, G2, and M phases and
divide
• If the cell does not receive the go-ahead signal, it will exit
the cycle, switching into a nondividing state called the G0
phase
Figure 12.17
G1 checkpoint
G0
G1
G1
Without go-ahead signal,
cell enters G0.
G1
S
M
With go-ahead signal,
cell continues cell cycle.
(a) G1 checkpoint
G2
G1
G1
M
G2
M
G2
M checkpoint
Anaphase
Prometaphase
Without full chromosome
attachment, stop signal is
received.
(b) M checkpoint
G2
checkpoint
Metaphase
With full chromosome
attachment, go-ahead signal
is received.
The G1 or G0 Checkpoint
• For many cells, the G1
checkpoint seems to be the
most important one
• If a cell receives a go-ahead
signal at the G1 checkpoint,
it will usually complete the
S, G2, and M phases and
divide
• If the cell does not receive
the go-ahead signal, it will
exit the cycle, switching into
a non dividing state called
the G0 phase
• Most human cells are
in G0 phase are
nerve, liver and
muscle cells.
© 2014 Pearson Education, Inc.
Figure 12.17
Stop and Go Signs: Internal and External
Signals at the Checkpoints
• An example of an internal signal is that anaphase is
delayed until all of the chromosomes are attached to the
spindle at the metaphase plate
• Some external signals are growth factors, proteins
released by certain cells that stimulate other cells to divide
– For example, platelet-derived growth factor (PDGF) stimulates the
division of human fibroblast cells in culture
• Another example of external signals is density-dependent
inhibition, in which crowded cells stop dividing
• Most animal cells also exhibit anchorage dependence, in
which they must be attached to a substratum in order to
divide
• Density-dependent inhibition and anchorage dependence
check the growth of cells at an optimal density
• Cancer cells exhibit neither type of regulation of their
division
© 2014 Pearson Education, Inc.
Figure 12.18
Scalpels
1 A sample of
human connective
tissue is cut
up into small
pieces.
Petri
dish
2 Enzymes digest
the extracellular
matrix, resulting
in a suspension of
free fibroblasts.
3 Cells are transferred
to culture vessels.
4 PDGF is added
10 µm
to half the
vessels.
Without PDGF
With PDGF
Cultured fibroblasts (SEM)
Figure 12.19
Anchorage dependence: cells
require a surface for division
Density-dependent inhibition:
cells form a single layer
Density-dependent inhibition:
cells divide to fill a gap and
then stop
20 µm
(a) Normal mammalian cells
20 µm
(b) Cancer cells
Loss of Cell Cycle Controls in Cancer Cells
• Cancer cells do not respond normally to
the body’s control mechanisms
– Cancer cells exhibit neither density-dependent
inhibition nor anchorage dependence
• Cancer cells may not need growth
factors to grow and divide:
– They may make their own growth factors
– They may convey a growth factor’s signal
without the presence of the growth factor
– They may have an abnormal cell cycle control
system
© 2014 Pearson Education, Inc.
Transformation
• A normal cell is converted to a cancerous cell by a process
called transformation.
• Cancer cells form tumors, masses of abnormal cells within
otherwise normal tissue.
• If abnormal cells remain at the original site, the lump is
called a benign tumor.
• Malignant tumors invade surrounding tissues and can
metastasize, exporting cancer cells to other parts of the
body, where they may form secondary tumors.
• Localized tumors may be treated with highenergy radiation, which damages the DNA in
the cancer cells
• To treat metastatic cancers, chemotherapies
that target the cell cycle may be used
© 2014 Pearson Education, Inc.
5 µm
Figure 12.20
Breast cancer cell
(colorized SEM)
Lymph
vessel
Metastatic
tumor
Tumor
Blood
vessel
Cancer
cell
Glandular
tissue
1 A tumor grows
from a single
cancer cell.
2 Cancer cells invade
neighboring tissue.
3 Cancer cells spread
through lymph and
blood vessels to other
parts of the body.
4 A small percentage
of cancer cells may
metastasize to
another part of the
body.
Recent advances in understanding the cell cycle and cell cycle signaling
have led to advances in breast cancer treatment
Breast cancer cells as shown above are analyzed DNA sequencing and
alternation of specific protein sequence associated with cancer. For example
20-25% of breast cancer tumors show abnormally high amounts of cell
surface receptor tyrosine kinase called HER2 and many show increase
in the number of estrogen receptor called ER molecules, that can trigger
cell division. Based on this physician do chemotherapy that blocks the function
of specific protein (Herceptin for HER2 and tamoxifen for ERs). Treatment
of these drugs led increased survival rate and fewer cancer recurrences.
• Recent advances in understanding the cell
cycle and cell cycle signaling have led to
advances in cancer treatment
• Coupled with the ability to sequence the
DNA of cells in a particular tumor,
treatments are becoming more
“personalized”.
Figure 12.UN02
S
G1
Cytokinesis
G2
Mitosis
MITOTIC (M)
PHASE
Prophase
Telophase
and
Cytokinesis
Prometaphase
Anaphase
Metaphase
Figure 12.UN03