Gitam

Department of biotechnology
Msc semester 1 2021 -2023
Cell biology and genetics assignment
Subject code : 707
Topic: cell cycle and its regulation
Name :p. navya rajasree
Roll no:vp21btsc0100045

Submitted to : prabhakar sir

Introduction

The division of all cells must be carefully regulated and coordinated with both cell growth
and DNA replication in order to ensure the formation of progeny cells containing intact
genomes. In eukaryotic cells, progression through the cell cycle is controlled by a series of
protein kinases that have been conserved from yeasts to mammals. In higher eukaryotes,
this cell cycle machinery is itself regulated by the growth factors that control cell
proliferation, allowing the division of individual cells to be coordinated with the needs of the
organism as a whole.

Stages of Cell Cycle

A cell spends most of its time in what is called interphase, and during this time it grows,
replicates as chromosome , and prepares for cell division. The cell then leaves interphase,
undergoes mitosis, and completes its division. The resulting cells, known as daughter cells,
each enter their own interphase and begin a new round of the cell cycle.
 Interphase Stage
The interphase stage of the cell cycle includes three distinctive parts: the G1 phase, the S
phase, and the G2 phase. 
G1 is the stage where the cell is preparing to divide. To do this, it then moves into the S
phase where the cell copies all the DNA. So, S stands for DNA synthesis. After the DNA is
copied and there’s a complete extra set of all the genetic material, the cell moves into the
G2 stage, where it organizes and condenses the genetic material, or starts to condense the
genetic material, and prepares to divide.
Mitosis Stage
The next stage is M. M stands for mitosis. This is where the cell actually partitions the two
copies of the genetic material into the two daughter cells. After M phase completes, cell
division occurs and two cells are left, and the cell cycle can begin again.
Mitosis is a continuous process, but for convenience in denoting which portion of the
process is taking place, scientists divide mitosis into a series of phases:-
 (1) Karyokinesis (Nuclear division) – divided into 4 sub-phases:-
(a) Prophase
Mitosis begins with the condensing of the chromatin to form chromosomes in the phase
called prophase. Two copies of each chromosome exist; each one is a chromatid. Two
chromatids are joined to one another at a region called the centromere. As prophase
unfolds, the chromatids become visible in pairs (called sister chromatids), the spindle fibers
form, the nucleoli disappear, and the nuclear envelope dissolves.
 In animal cells during prophase, microscopic bodies called centrioles begin to migrate to
opposite sides of the cell. When the centrioles reach the poles of the cell, they produce and
are then surrounded by a series of radiating microtubules called an aster. Centrioles and
asters are not present in most plant or fungal cells.
 As prophase continues, the chromatids attach to spindle fibers that extend out from
opposite poles of the cell. The spindle fibers attach at the region of the centromere at a
structure called the kinetochore, an area of protein in the centromere region. Eventually, all
pairs of chromatids reach the center of the cell, a region called the equatorial plate.
(b) Metaphase
Metaphase is the stage of mitosis in which the pairs of chromatids line up on the equatorial
plate. This region is also called the metaphase plate. In a human cell, 92 chromosomes in 46
pairs align at the equatorial plate. Each pair is connected at the centromere, where the
spindle fiber is attached (more specifically at the kinetochore).
(c) Anaphase
At the beginning of anaphase, the sister chromatids move apart from one another. The
chromatids are called chromosomes after the separation. Each chromosome is attached to a
spindle fiber, and the members of each chromosome pair are drawn to opposite poles of
the cell by the spindle fibers. During anaphase, the chromosomes can be seen moving. They
take on a rough V shape because of their midregion attachment to the spindle fibers. The
movement toward the poles is accomplished by several mechanisms, such as an elongation
of the spindle fibers, which results in pushing the poles apart.
 The result of anaphase is an equal separation and distribution of the chromosomes. In
human cells, a total of 46 chromosomes move to each pole as the process of mitosis
continues.
(d) Telophase
In telophase, the chromosomes finally arrive at the opposite poles of the cell. The distinct
chromosomes begin to fade from sight as masses of chromatin are formed again. The events
of telophase are essentially the reverse of those in prophase. The spindle is dismantled and
its amino acids are recycled, the nucleoli reappear, and the nuclear envelope is reformed.
(2) Cytokinesis (Division of Cytoplasm)
 Leading to halving of that genome, passing into daughter cells.
Cytokinesis is the process in which the cytoplasm divides and two separate cells form. Note
that cytokinesis is separate from the four stages of mitosis. In animal cells, cytokinesis
begins with the formation of a cleavage furrow in the center of the cell. With the formation
of the furrow, the cell membrane begins to pinch into the cytoplasm, and the formation of
two cells begins. This process is often referred to as cell cleavage . Microfilaments contract
during cleavage and assist the division of the cell into two daughter cells.
 In plant cells, cytokinesis occurs by a different process because a rigid cell wall is involved.
Cleavage does not take place in plant cells. Rather, a new cell wall is assembled at the center
of the cell, beginning with vesicles formed from golgi. As the vesicles join, they form a
double membrane called the cell plate. The cell plate forms in the middle of the cytoplasm
and grows outward to fuse with the cell membrane. The cell plate separates the two
daughter cells. As cell wall material is laid down, the two cells move apart from one another
to yield two new daughter cells.
Mitosis serves several functions in living cells. In many simple organisms, it is the method for
asexual reproduction (for example, in the cells of a fungus). In multicellular organisms,
mitosis allows the entire organism to grow by forming new cells and replacing older cells. In
certain species, mitosis is used to heal wounds or regenerate body parts. It is the universal
process for cell division in eukaryotic cells.
We can summarized the functions of each phase as following:
G1 phase
 Cell increases in size
 Cellular contents are duplicated
S phase
 DNA replication
 Each of the 46 chromosomes (23 pairs) is replicated by the cell
G2 phase
 Cell grows more
 Organelles and proteins develop in preparation for cell division
M phase
 Mitosis followed by cytokinesis (cell separation)
 Formation of two identical daughter cells
G0 phase
While some cells are constantly dividing, some cell types are quiescent. These cells exit G1
and enter a resting state called G0. In G0, a cell is performing its function without actively
preparing to divide. G0 is a permanent state for some cells, while others may restart division
if they get the right signals.

Cell Cycle Checkpoints

Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell
cycle. The cell can’t proceed to the next phase until checkpoint requirements have been
met.
There are three main checkpoints:
1) G1/S checkpoint (before cell enters S phase)
2) G2/M checkpoint (after S phase)
3) APC/C checkpoint (during mitosis)

The controls discussed in the previous section regulate cell cycle progression in response to
cell size and extracellular signals, such as nutrients and growth factors. ln addition, the
events that take place during different stages of the cell cycle must be coordinated with one
another so that they occur in the appropriate order. For example, it is critically important
that they occur in the appropriate order. For example, it is critically important that the cell
not begin mitosis until replication of the genome has been completed. The alternative
would be a catastrophic cell division in which the daughter cells failed to inherit complete
copies of the genetic material. [n most cells, this coordination between different phases of
the cell cycle is dependent on a series of cell cycle checkpoints that prevent entry into the
next phase of the cell cycle until the events of the preceding phase have been completed.
Several cell cycle checkpoints function to ensure that incomplete or damaged chromosomes
are not replicated and passed on to daughter cells. These checkpoints sense unreplicated or
damaged DNA and coordinate further cell cycle progression with the completion of DNA
replication or repair. For example, the checkpoint in G2 prevents the initiation of mitosis
until DNA replication is completed. This G2 checkpoint senses unreplicated DNA, which
generates a signal that leads to cell cycle arrest. Operation of the G2 checkpoint therefore
prevents the initiation of M phase before completion of S phase, so cells remain in G2 until
the genome has been completely replicated. Only then is the inhibition of G2 progression
relieved, allowing the cell to initiate mitosis and distribute the completely replica ted
chromosomes to daughter cells. In addition to sensing unreplicated DNA, the G2 checkpoint
senses DNA damage, such as that resulting from irradiation. If DNA damage is detected,
arrest at the checkpoint allows time for the damage to be repaired, rather than being
passed on to daughter cells.
DNA damage not only arrests the cell cycle in G2 but also at checkpoints in G1 and S phase.
Arrest at the G1 checkpoint allows repair of the damage to take place before the cell enters
S phase, where the damaged DNA would be replicated. The S-phase checkpoint provides
continual monitoring of the integrity of DNA to ensure that damaged DNA is repaired before
it is replicated. In addition, the S-phase checkpoint provides a quality control monitor to
promote the repair of any errors that occur during DNA replication, such as the
incorporation of incorrect bases or incomplete replication of segments of DNA. Cell cycle
arrest at the G1, S, and G2 checkpoints is mediated by two related protein kinases,
designated ATM and ATR, that recognize damaged or unreplicated DNA and are activated in
response to DNA damage. ATM and ATR then activate a signaling pathway that leads not
only to cell cycle arrest, but also to the activation of DNA repair and, in some cases,
programmed cell death. The importance of checkpoint regulation is emphasized by the fact
that these proteins were initially identified because mutations in the gene encoding ATM
are responsible for the disease ataxia telangiectasia, which results in defects in the nervous
and immune systems as well as a high frequency of cancer in affected individuals.

Cell cycle regulation

Three initially distinct experimental approaches contributed to identification of the key

molecules responsible for cell cycle regulation. The first of these avenues of investigation
originated with studies of frog oocytes . These oocytes are arrested in the G2 phase of the
cell cycle until hormonal stimulation triggers their entry into theM phase of meiosis . In
1971, two independent teams of researchers (Yoshio Masui and Clement Markert, as well as
Dennis Smith and Robert Ecker) found that oocytes arrested in G2 could be induced to enter
M phase by microinjection of cytoplasm from oocytes that had been hormonally stimulated.
It thus appeared that a cytoplasmic factor present in hormonetreated oocytes was sufficient
to trigger the transition from G2 toM in oocytes that had not been exposed to hormone.
Because the entry of oocytes into meiosis is frequently referred to as oocyte maturation,
this cytoplasmic factor was called maturation promoting factor (MPF). Further studies
showed, however, that the activity of MPF is not restricted to the entry of oocytt::s into
meiosis. To the contrary, MPF is abo present in somatic cells, where it induces entry into M
phase of the mitotic cycle. Rather than being specific to oocytes, MPF thus appeared to act
as a general regulator of the transition from G2 to M. The second approach to
understanding cell cycle regulation was the genetic analysis of yeasts, pioneered by Lee
Hartwell and his colleagues in the early 1970s. Studying the budding yeast Saccharomyces
cerevisiae, these investigators identified temperature-sensitive mutants that were defective
in cell cycle progression. The key characteristic of these mutants (called cdc for cell division
cycle mutants) was that they underwent growth arrest at specific points in the cell cycle. For
example, a particularly important mutant designated cdc28 caused the cell cycle to arrest at
START, indicating that the Cdc28 protein is required for passage through this critical
regulattory point in G1 . A similar collection of cell cycle mutants was isolated in the fission
yeast Schizosaccharomyces pombe by Paul Nurse and his collaborators. These mutants
included cdc2, which arrests the S. pombe cell cycle both in G1 and at the G2 toM transition
(the major regulatory point in fission yeast). Comparative analysis showed that S. cerevisiae
cdc28 and S. pombe cdc2 are functionally homologous genes, which are required for
passage through START as well as for entry into mitosis in both species of yeasts. Further
studies demonstrated that cdc2 and cdc28 encoded a protein kinase-the first indication of
the prominent role of protein phosphorylation in regulating the cell cycle. In addition,
related genes were identified in other eukaryotes, including humans. The protein kinase
encoded by the yeast cdc2 and cdc28 genes has since been shown to be a conserved cell
cycle regulator in all eukaryotes, which is known as Cdk1. The third line of investigation that
eventually converged with the identification of MPF and yeast genetics emanated from
studies of protein synthesis in early sea urchin embryos. Following fertilization, these
embryos go through a series of rapid cell divisions. Intriguingly, studies with protein
synthesis inhibitors had revealed that entry into M phase of these embryonic cell cycles
requires new protein synthesis. In 1983, Tim Hunt and his colleagues identified two proteins
that display a periodic pattern of accumulation and degradation in sea urchin and clam
embryos. These proteins accumulate throughout interphase and are then rapidly degraded
toward the end of each mitosis . Hunt called these proteins cyclins (the two proteins were
designated cyclin A and cyclin B) and suggested that they might function to induce mitosis,
with their periodic accumulation and destruction controlling entry and exit from M phase.
Direct support for such a role of cyclins was provided in 1986, when Joan Ruderman and her
colleagues showed that microinjection of cyclin A into frog oocytes is sufficient to trigger the
G2 toM transition.
These initially independent approaches converged dramatically in 1988 when MPF was
purified from frog eggs in the laboratory of James Maller. Molecular characterization of MPF
in several laboratories then showed that this conserved regulator of the cell cycle is
composed of two key subunits: Cdkl and cyclin B . Cyclin B is a regulatory subunit required
for catalytic activity of the Cdkl protein kinase, consistent with the notion that MPF activity
is controlled by the periodic accumulation and degradation of cyclin B during cell cycle
progression. A variety of further studies have confirmed this role of cyclin B, as well as
demonstrating the regulation of MPF by phosphorylation and dephosphorylation of Cdkl . In
mammalian cells, cyclin B is synthesized and forms complexes with Cdkl during G2. As these
complexes form, Cdkl is phosphorylated at two critical regulatory positions. One of these
phosphorylations occurs on threonine-161 and is required for Cdk1 kinase activity. The
second is a phosphorylation of tyrosine-1S and of the adjacent threonine-1-1 in vertebrates.
Phosphorylation of tyrosine-IS, catalyzed by a protein kinase called Wee1, inhibits Cdk1
activity and leads to the accumulation of inactive Cdkl/cyclin B complexes throughout G2.
The transition from G2 toM is then brought about by activation of the Cdk1/cyclin B
complex as a result of dephosphorylation of threonine-14 and tyrosine-IS by a protein
phosphatase called Cdc2SC.
Once activated, the Cdkl protein kinase phosphorylates a variety of target proteins that
initiate the events of M phase, which are discussed later in this chapter. In addition, Cdkl
activity triggers the degradation of cyclin B, which occurs as a result of ubiquitin-mediated
proteolysis. This proteolytic destruction of cyclin B then inactivates Cdkl, leading the cell to
exit mitosis, undergo cytokinesis, and return to interphase.
 Mechanism of cell cycle regulation by CDKs activation
Multiple mechanisms ensure that CDKs are active in the right stage of the cell cycle. CDK
activity is regulated by multiple mechanisms:
1. Regulating the cyclin levels
2. Action of CDK-activating kinase (CAK)
3. Inhibitory phosphorylations on CDK
4. Action of CDK Inhibitors
5. Regulation of Cyclin Levels
The timely activation of CDKs depends, in part, on the presence of the appropriate cyclins in
the cell cycle stage where they are needed.
Cells utilize multiple mechanisms to restrict cyclins to the appropriate cell cycle stage and to
keep them at the right concentration.
 1.Regulation of Cyclin Mechanisms:
 Transcriptional control of cyclin genes.
 Degradation of cyclins.
 Transcriptional control of the cyclin subunits is one mechanism that ensures proper
temporal expression of the cyclins.
 Degradation of cyclins.
The most important regulatory control that restricts cyclins to the appropriate cell cycle
stage is ubiquitin-mediated protein degradation.
 Cyclins are degraded through the action of two different ubiquitin-proteins:
1. a) SCF (Skp1,Cullin & f-box proteins)
2. b) APC/C (anaphase-promoting complex or cydosome)
SCF controls the G1-S phase transition by degrading G1 cyclins (Cyclin D) The APC/C
degrades S phase and mitotic cyclins, thereby promoting the exit from mitosis.
Regulating the levels of cyclins is not the only mechanism that controls CDK activity.
 Activating and inhibitory phosphorylation events on the CDK subunit and presence of
inhibitors are essential to the control cyclin-CDK activity.
2. CDK-activating kinase (CAK)
 Phosphorylation of a threonine residue is required for CDK activity. This phosphorylation is
mediated by the CDK-activating kinase (CAK). CAK activity is constant throughout the cell
cycle and phosphorylates the CDK as soon as a cyclin-CDK complex is formed.
3. Inhibitory phosphorylations on CDK
Inhibitory phosphorylation on CDK plays a critical role in controlling CDK activity. A kinase
called “Weel” brings about this inhibitory phosphorylation.
4. CDK Inhibitors (CKIs)
CDK inhibitors control Cyclin-CDK activity
Family of proteins known as CDK inhibitors or CKIs, that directly bind to the cyclin-CDK
complex and inhibit its
activity.
These proteins play an especially important role in the regulation of the G1-S phase
transition (entry into the cell cycle).the genes encoding these CKIs are often found mutated
in human cancers CKIs involved in regulating S phase and mitotic CDKs are all essential to
prevent premature activation of S phase and M phase CDKs.
Inhibitors of G1 CDKs play an essential role in mediating a G1 arrest in response to
proliferation inhibitory signals.
 A class of CKIs called INK4s interact only with the G1 CDKs. Binding of INK4s to CDK4 and
CDK6 blocks their interaction with cyclin D and hence their protein kinase activity.
A second class of CKIs found in metazoan cells consists of three proteins; p21 p27 and p57.
 p53 is a TF, which stimulates the expression of p21, p27 and p57 genes.
 The p21 protein inhibits cyclin/CDK protein complexes that are needed to progress from
the G1 phase of the cell cycle to the S phase. CKIs regulating G1 CDKs play a critical role in
preventing tumor formation.
CDK Inhibitors
 One of important cyclins that play a vital role in cell cycle regulation is  cyclin-dependent
kinase inhibitor (CKI) is a protein that interacts with a cyclin-CDK complex to block kinase
activity, usually during G1 or in response to signals from the environment or from damaged
DNA. In animal cells, there are two major CKI families: the INK4 family and
the CIP/KIP family. The INK4 family proteins are strictly inhibitory and bind CDK monomers.
Crystal structures of CDK6-INK4 complexes show that INK4 binding twists the CDK to distort
cyclin binding and kinase activity. The CIP/KIP family proteins bind both the cyclin and the
CDK of a complex and can be inhibitory or activating.

1) G1/S checkpoint (before cell enters S phase):

► Checks for cell size
► Checks for nutrients
► Checks for DNA damage
► Checks for all the preparations (all proteins, ATP etc. requires in S phase)
► Checks whether S phase Cyclins and Cdk complex is activated to initiate DNA replication.
Then the cell passes to the next S phase.
2) G2/M checkpoint (after S phase):
► Checks for proper DNA replication
► Checks for all the preparations (all proteins, ATP etc. required in M phase)
► Checks for Tubulin synthesis
► Checks whether M phase Cyclins and Cdk complex is activated to initiate mitosis
Then the cell passes to the next M phase.
 All the checkpoints require the services of a complex of proteins. The levels of these
proteins are increased in damaged cells. They allow time to prepair DNA by blocking the cell
cycle.
P53 is a protein which senses DNA damage and can halt progression of the cell cycle in G1
phase by blocking the activity of Cdk2 until damage can be repaired. If the damage is so
severe that it can’t be repaired, then the cell destructs itself by apoptosis. In case of damage
to DNA after S phase, the action of Cdk1 is inhibited, thus stopping progression of the cell
from G2 to mitosis.
Fast Facts about Cell Cycle
 Cells in S phase have more DNA than cells in G1 phase. Cells in G2 will have approximately
twice DNA content as cells in G1. Thus, they will take up proportionately more dye. The
suspension of cells is then aspirated into a flow cell. Cells, surrounded by a narrow fluid
stream, pass one by one through a focused laser beam. The light is either scattered or
absorbed when it strikes a cell.
 The Nobel Prize in Physiology or Medicine 2001 was awarded jointly to Leland H. Hartwell,
Tim Hunt and Sir Paul M. Nurse for their discoveries of key regulators of the cell cycle.
 Absorbed light of the appropriate wavelength is reemitted as fluorescence. This reflects
the internal structure of the cell and its size and shape. Fluorescence scatter signals are
detected, amplified and analyzed by a series of photodiodes and a computer system.
 

 The end result is quantitative information about every cell analyzed. Large numbers of
cells are analyzed in a short period of time (>1,000/sec). This gives the advantage of
creating statistically valid information about cell populations.
 

The potential applications of flow cytometry include the detection and measurement of:
1. Cell cycle: Reliable assessment of cells in G0/G1 phase versus S phase, G2, or polyploidy,
including analysis of cell proliferation and activation.
2. Cell viability/apoptosis.
3. Identification and characterization of distinct subsets of cells within a heterogeneous
sample.
4. Protein expression.
5. Protein post translational modifications.
6. RNA, including IncRNA, miRNA, and mRNA transcripts.