Please write your answer briefly and concisely.

1.      Cell division plays important role in growth and development of organisms.

a.      why each organism needs to coordinate cell division?

Coordination of cell division in multicellular organisms is critical for normal growth,

development and maintenance. Cells have an upper and lower size limit. If it was too small it
would not contain all organelles. It can't be too big because of the ratio of the cell surface area
to volume. An increase in size can result in a surface area too small for the adequate exchange
of materials. Some cells must be large for a purpose to maintain a suitable size, growth and
division must be coordinated, if a cell doubles in size the daughter cells will be too big or too
small, this will happen with each generation and the cell will die. Coordination of cell growth and
division is important for cells to stay the same size from generation to generation.

Control of cell division is important because too much cell division can cause mutation
and overgrowth of cells resulting in cancerous grouts.

To conserve energy and to divide at appropriate times

Coordinating the timing of cell division and rates of cell division so that all cells are not
on the same cycle then you wouldn’t have cell available for functions at all times

b.      how frequent cell division occurs?

About 78% of a cell’s life is spent in Interphase, growing and preparing itself for cell
division. Interphase itself contains three subphases. Immediately after cell division, a newly-
formed cell enters the Gap 1 or G1 portion of Interphase. During G1, cells actively grow and, in
many cases, differentiate to perform specific functions. At this stage, the cell is sensitive to
internal and external signals to determine whether to divide or not. Some cells, such as neurons
do not proceed to cell division after differentiation and remain in G1 until they die. Once the cell
meets criteria to proceed to cell division, it enters the synthesis or S stage of Interphase, during
which the cell replicates its entire genome. Next, the cell enters the Gap 2 or G2 phase to
synthesize all the necessary proteins for cell division. This is followed by the cell division phase

c.      how regulation of cell division ? is it the same for each cell type, tissues and organs?

Control of cell division is important because too much cell division can cause mutation
and overgrowth of cells resulting in cancerous growths. A multicellular organism uses:

Internal Controls to coordinate and control this division. A multicellular organism must have
MPF, which is Cdk and Cyclin put together. This turns on other proteins for mitosis.

External Controls to coordinate and control this division: Growth factors -- coordination
between cells, protein signals released by body cells that stimulate other cells to divide--
Positional Inhibition--Animal Cells must be anchored (attached to a substrate)to divide -- Also
they cannot be too crowded ("Density Dependant Inhibition")- Crowded cells stop dividing. Each
 cell binds a bit of growth factor so that there is not enough activator left to trigger division in
any one cell.

2.       There is signal to control cell cycle i.e., cyclin, cdks, cdks-cyclin complex. Please explain the role
each of them in relation to cell cycle regulation. Explaining with figure may be helpful.

Cyclins are among the most important core cell cycle regulators. Cyclins are a group of related
proteins, and there are four basic types found in humans and most other eukaryotes: G1 cyclins,
G1/S cyclins, S cyclins, and M cyclins.

Cyclin-dependent kinases

In order to drive the cell cycle forward, a cyclin must activate or inactivate many target proteins
inside of the cell. Cyclins drive the events of the cell cycle by partnering with a family of enzymes
called the cyclin-dependent kinases (Cdks). A lone Cdk is inactive, but the binding of a cyclin
activates it, making it a functional enzyme and allowing it to modify target proteins.
3.      What is the limit for cell growth?

a.      How to regulate cell growth?

Cell division is controlled not only by extracellular mitogens but also by intracellular mechanisms
that can limit cell proliferation. Many animal precursor cells, for example, divide a limited
number of times before they stop and terminally differentiate into permanently arrested,
specialized cells. Although the stopping mechanisms are poorly understood, a progressive
increase in CKI proteins probably contributes in some cases

b.      What is internal regulators?

Internal regulators are proteins that regulate cell growth from inside of the cell. Internal
regulators are special proteins that permit the cell cycle to continue only when certain
conditions have been met inside the cell. For example mitosis can't proceed unless the DNA has
been copied. Thus it allows the cell cycle to proceed only when certain processes have
happened inside the cell

c.      What is external regulators?

External regulators are proteins outside of the cell that also regulate cell growth. External
regulators that respond to events outside the cell and speed up or slow down the cell cycle.
What is an example of an external regulator? Proto-oncogenes that promote cell division and
tumor suppressors that inhibit cell division. With both of these off cancer cells form.
 d.      What is uncontrolled cell growth?

Abnormal regulation of the cell cycle can lead to the over proliferation of cells and an
accumulation of abnormal cell numbers. Cancer cells arise from one cell that becomes damaged,
and when divided, the damage is passed on to the daughter cell and again to the granddaughter
cells and so on. Such uncontrolled, abnormal growth of cells is a defining characteristic of cancer

4.      Is there any mechanism to control cell cycle?

a.      what is the checkpoint? What is the function of check point?

A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal and
external cues and "decides" whether or not to move forward with division.

This regulation makes sure that cells don't divide under unfavorable conditions (for instance,
when their DNA is damaged, or when there isn't room for more cells in a tissue or organ).

There are a number of checkpoints, but the three most important ones are:

 The G1 checkpoint, at the G1/s transition.

 The G2 checkpoint, at the G2 /M transition.
 The spindle checkpoint, at the transition from metaphase to anaphase.

b.      how do cell know when to divide?

In cell division, the cell that is dividing is called the "parent" cell. The parent cell divides
into two "daughter" cells. The process then repeats in what is called the cell cycle.  

Cells regulate their division by communicating with each other using chemical signals
from special proteins called cyclins. These signals act like switches to tell cells when to start
dividing and later when to stop dividing. It is important for cells to divide so you can grow and so
your cuts heal. It is also important for cells to stop dividing at the right time.  If a cell can not
stop dividing when it is supposed to stop, this can lead to a disease called cancer

Some cells, like skin cells, are constantly dividing. We need to continuously make new
skin cells to replace the skin cells we lose. Did you know we lose 30,000 to 40,000 dead skin cells
every minute? That means we lose around 50 million cells every day.  This is a lot of skin cells to
replace, making cell division in skin cells is so important. Other cells, like nerve and brain cells,
divide much less often

c.      How relation between cancer and cell growth?

The fundamental abnormality resulting in the development of cancer is the continual

unregulated proliferation of cancer cells. Rather than responding appropriately to the signals
that control normal cell behavior, cancer cells grow and divide in an uncontrolled manner,
invading normal tissues and organs and eventually spreading throughout the body
 The generalized loss of growth control exhibited by cancer cells is the net result of
accumulated abnormalities in multiple cell regulatory systems and is reflected in several aspects
of cell behavior that distinguish cancer cells from their normal counterparts

5.      Failure in controlling cell growth could possibly create cancer cells. How is the cancer growth?

The fundamental abnormality resulting in the development of cancer is the continual

unregulated proliferation of cancer cells. Rather than responding appropriately to the signals
that control normal cell behavior, cancer cells grow and divide in an uncontrolled manner,
invading normal tissues and organs and eventually spreading throughout the body

The generalized loss of growth control exhibited by cancer cells is the net result of accumulated
abnormalities in multiple cell regulatory systems and is reflected in several aspects of cell
behavior that distinguish cancer cells from their normal counterparts

6.       In our cell, gene ‘p53’ has been known to play essential role in cell division. What is the ‘p53’
gene? And what is its role?

The p53 protein is located in the nucleus of cells throughout the body, where it attaches (binds)
directly to DNA. When the DNA in a cell becomes damaged by agents such as toxic chemicals,
radiation, or ultraviolet (UV) rays from sunlight, this protein plays a critical role in determining
whether the DNA will be repaired or the damaged cell will self-destruct (undergo apoptosis). If
the DNA can be repaired, p53 activates other genes to fix the damage. If the DNA cannot be
repaired, this protein prevents the cell from dividing and signals it to undergo apoptosis. By
stopping cells with mutated or damaged DNA from dividing, p53 helps prevent the development
of tumors.

7.       a. What is cell communication (in terms of cell biology)?

The communication network between one cell and another will result in a coordination in order
to regulate growth, osmoregulation, reproduction and other things in various tissues and
organs.

In unicellular organisms, all activities that support cell life are carried out by the cell itself

In multicellular organisms all activities are not carried out alone there is cooperation between
these cells.

b. What is the different between local and long distance signaling? Please explain with
mentioning some examples.

Short-distance signaling is a type of communication whereby a signaling molecule acts

on a target cell located close to the signaling cell. One type of short-distance signaling is also
 known as paracrine signaling, which is when signal molecules only diffuse through a short
extracellular space before they reach the target cell

Long-distance signaling is a type of communication whereby a signaling molecule acts

on a target cell far from the signaling cell. Long-distance signaling is also called endocrine
signaling.

8.      There are three steps of cell signaling. Please explain each step with sufficient information.

1. Reception – detection of message by a receptor protein on a target cell

2. Transduction – receptor changes and initiates a cascade of events

3. Response – activation of target

9.      What is apoptosis? Why is it important? How is it controlled? Explain in step by step basis.

A mechanism that allows cells to self-destruct when stimulated by the appropriate

trigger. Apoptosis can be triggered by mild cellular injury and by various factors internal or
external to the cell; the damaged cells are then disposed of in an orderly fashion

Apoptosis is a regulated cellular suicide mechanism characterized by nuclear

condensation, cell shrinkage, membrane blebbing, and DNA fragmentation. Caspases, a family of
cysteine proteases, are the central regulators of apoptosis. Initiator caspases (including caspase-
2, -8, -9, -10, -11, and -12) are closely coupled to pro-apoptotic signals. Once activated, these
caspases cleave and activate downstream effector caspases (including caspase-3, -6, and -7),
which in turn execute apoptosis by cleaving cellular proteins following specific Asp residues.
 Activation of Fas and TNFR by FasL and TNF, respectively, leads to the activation of
caspase-8 and -10.

DNA damage induces the expression of PIDD, which binds to RAIDD and caspase-2 and
leads to the activation of caspase-2.

10.   Academic paper entitled ”Application of CRISPR Genome Engineering in Cell Biology”
(Wang and Qi, 2016) is attached. The paper is very interesting to be learned and studied.
Please make a short resume regarding on the application, overview about the technique
(CRISPR), and other interesting notes.

Recent advances in genome engineering are starting a revolution in biological research

and translational applications. The CRISPR-associated RNA-guided endonuclease Cas9 and its
variants enable diverse manipulations of genome function.

the development of Cas9 tools for a variety of applications in cell biology research,
including the study of functional genomics, the creation of transgenic animal models, and
genomic imaging. Novel genome engineering methods offer a new avenue to understand the
causality between genome and phenotype, thus promising a fuller understanding of cell biology.

CRISPR/Cas9 technology has revolutionized cell biology research. The system is versatile,
enabling diverse types of genome engineering approaches. While most of the work has used
Cas9- mediated knockout or dCas9-mediated repression and activation to study gene function,
we expect expansion of these tools to study the epigenome and 3-dimensional chromosomal
organization in greater detail in the future. Furthermore, studies have used CRISPR to model
complex genomic rearrangements in vitro and in vivo, which brought breakthroughs in studying
chromosomal translocations. Most research has been performed in cell lines, and future work
related to the interrogation of cellular functions should be carried out in primary cells derived
from animals or human patients or in vivo using relevant animal models.

CRISPR/Cas9 is emerging as a major genome-manipulation tool for research and

therapeutics, yet there are remaining challenges to improve its specificity, efficiency, and utility
(see Outstanding Questions). One major concern is the off-target effects, since Cas9 can tolerate
mismatches between sgRNA and target DNA. Methods have been developed to profile the off-
target effects such as GUIDE-seq . To improve specificity, several strategies have been
developed including using paired nickase variants of Cas9, paired dCas9-FokI nucleases ,
truncated sgRNAs ((17 to 18 bp) that are more sensitive to mismatches, and controlling acting
concentration of the Cas9/sgRNA complex . Using structure-guided protein engineering
approaches, a few work recently created S. pyogenes Cas9 variants with improved specificity.
For example, a high fidelity variant of Cas9 harboring designed alterations showed reduced non-
specific DNA contacts, while retaining robust on-target activities comparable to wild-type Cas9.
Combinations of these methods could provide a route to its ultimate use for gene therapy.