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CYTOGENETICS 1

REVIEW OF CELL ORGANELLES

During the 1950s, scientists developed the concept that all organisms may be classified as
prokaryotes or eukaryotes. The cells of all prokaryotes and eukaryotes possess two basic
features: a plasma membrane, also called a cell membrane, and cytoplasm. However, the cells of
prokaryotes are simpler than those of eukaryotes. For example, prokaryotic cells lack a nucleus,
while eukaryotic cells have a nucleus. Prokaryotic cells lack internal cellular bodies (organelles),
while eukaryotic cells possess them. Examples of prokaryotes are bacteria and archaea.
Examples of eukaryotes are protists, fungi, plants, and animals (everything except prokaryotes).

Prokaryote Structure

Most prokaryotic cells are much smaller than eukaryotic cells. Although they are tiny,
prokaryotic cells can be distinguished by their shapes. The most common shapes are helices,
spheres, and rods.
Plasma Membrane and Cell Wall
Like other cells, prokaryotic cells have a plasma membrane. It controls what enters and leaves
the cell. It is also the site of many metabolic reactions. For example, cellular respiration and
photosynthesis take place in the plasma membrane.
Most prokaryotes also have a cell wall. It lies just outside the plasma membrane. It gives strength
and rigidity to the cell. Bacteria and Archaea differ in the makeup of their cell wall. The cell wall
of Bacteria contains peptidoglycan, composed of sugars and amino acids. The cell wall of most
Archaea lacks peptidoglycan.
Cytoplasm and Cell Structures
Inside the plasma membrane of prokaryotic cells is the cytoplasm. It contains several structures,
including ribosomes, a cytoskeleton, and genetic material. Ribosomes are sites where proteins
are made. The cytoskeleton helps the cell keep its shape. The genetic material is usually a single
loop of DNA. There may also be small, circular pieces of DNA, called plasmids. The cytoplasm
may contain microcompartments as well. These are tiny structures enclosed by proteins. They
contain enzymes and are involved in metabolic processes.
Extracellular Structures

Many prokaryotes have an extra layer, called a capsule, outside the cell wall. The capsule
protects the cell from chemicals and from drying out. It also allows the cell to stick to surfaces
and to other cells. Because of this, many prokaryotes can form biofilms. A biofilm is a colony of
prokaryotes that is stuck to a surface such as a rock or a host’s tissues. The sticky plaque that
collects on your teeth between brushings is a biofilm. It consists of millions of bacteria.

Most prokaryotes also have long, thin protein structures called flagella (singular, flagellum).
They extend from the plasma membrane. Flagella help prokaryotes move. They spin around a
fixed base, causing the cell to roll and tumble.

Model of a prokaryotic cell

Eukaryote structure
Plasma membrane
The plasma membrane (also known as the cell membrane) is the outermost cell surface, which
separates the cell from the external environment. The plasma membrane is composed primarily
of proteins and lipids, especially phospholipids. The lipids occur in two layers (a bilayer).
Proteins embedded in the bilayer appear to float within the lipid, so the membrane is constantly
in flux. The membrane is therefore referred to as a fluid mosaic structure. Within the fluid
mosaic structure, proteins carry out most of the membrane’s functions.

Cytoplasm and organelles

The cytoplasm (or cytosol) is a semiliquid substance that composes the volume of a cell.
Essentially, cytoplasm is the gel-like material enclosed by the plasma membrane. Within the
cytoplasm of eukaryotic cells are several membrane-bound bodies called organelles (“little
organs”) that provide a specialized function within the cell.

One example of an organelle is the endoplasmic reticulum (ER). The ER is a series of

membranes extending throughout the cytoplasm of eukaryotic cells. In some places, the ER is
studded with submicroscopic bodies called ribosomes. This type of ER is called rough ER. In
other places, there are no ribosomes. This type of ER is called smooth ER. The rough ER is the
site of protein synthesis in a cell because it contains ribosomes; however, the smooth ER lacks
ribosomes and is responsible for producing lipids. Within the ribosomes, amino acids are bound
together to form proteins. Cisternae are spaces within the folds of the ER membranes.

Another organelle is the Golgi apparatus (also called Golgi body). The Golgi apparatus is a
series of flattened sacs, usually curled at the edges. In the Golgi body, the cell’s proteins and
lipids are processed and packaged before being sent to their destination. To accomplish this
function, the outermost sac of the Golgi body often bulges and breaks away to form drop-like
vesicles known as secretory vesicles.

An organelle called the lysosome is derived from the Golgi body. It is a drop-like sac of enzymes
in the cytoplasm. These enzymes are used for digestion within the cell. They break down
particles of food taken into the cell and make the products available for use; they also help break
down old cell organelles. Enzymes are also contained in a cytoplasmic body called the
peroxisome.
The components of an idealized eukaryotic cell. The diagram shows the relative sizes and
locations of the cell parts.

The organelle that releases quantities of energy to form adenosine triphosphate (ATP) is the
mitochondrion (the plural form is mitochondria). Because mitochondria are involved in energy
release and storage, they are called the “powerhouses of the cells.”

Green plant cells, for example, contain organelles known as chloroplasts, which function in the
process of photosynthesis. Within chloroplasts, energy from the sun is absorbed and transformed
into the energy of carbohydrate molecules. Plant cells specialized for photosynthesis contain
large numbers of chloroplasts, which are green because the chlorophyll pigments within the
chloroplasts are green. Leaves of a plant contain numerous chloroplasts. Plant cells not
specializing in photosynthesis (for example, root cells) are not green.

An organelle found in mature plant cells is a large, fluid-filled central vacuole. The vacuole may
occupy more than 75 percent of the plant cell. In the vacuole, the plant stores nutrients, as well as
toxic wastes. Pressure within the growing vacuole may cause the cell to swell.

The cytoskeleton is an interconnected system of fibers, threads, and interwoven molecules that
give structure to the cell. The main components of the cytoskeleton are microtubules,
microfilaments, and intermediate filaments. All are assembled from subunits of protein.
The centriole organelle is a cylinder-like structure that occurs in pairs. Centrioles function in cell
division.

Many cells have specialized cytoskeletal structures called flagella and cilia. Flagella are long,
hair-like organelles that extend from the cell, permitting it to move. In prokaryotic cells, such as
bacteria, the flagella rotate like the propeller of a motorboat. In eukaryotic cells, such as certain
protozoa and sperm cells, the flagella whip about and propel the cell. Cilia are shorter and more
numerous than flagella. In moving cells, the cilia wave in unison and move the cell forward.
Paramecium is a well-known ciliated protozoan. Cilia are also found on the surface of several
types of cells, such as those that line the human respiratory tract.

Cell wall

Many kinds of prokaryotes and eukaryotes contain a structure outside the cell membrane called
the cell wall. Among the eukaryotes, some protists, and all fungi and plants, have cell walls. Cell
walls are not identical in these organisms, however. In fungi, the cell wall contains a
polysaccharide called chitin. Plant cells, in contrast, have no chitin; their cell walls are composed
exclusively of the polysaccharide cellulose. Cell walls provide support and help cells resist
mechanical pressures, but they are not solid, so materials are able to pass through rather easily.
Cell walls are not selective devices, as plasma membranes are.

Differences between prokaryotic and eukaryotic cells

Prokaryotic Cell Eukaryotic cell
Size is 0.1- 5.0 um Size is 5-100 um
Nucleus is absent Nucleus is present
Membrane-bound nucleus absent. Membrane-bound Nucleus is present.
One chromosome is present, but not true More than one number of chromosomes is present.
chromosome plastids
Unicellular Multicellular
Lysosomes and Peroxisomes absent Lysosomes and Peroxisomes present
Microtubules absent Microtubules present
Endoplasmic reticulum absent Endoplasmic reticulum present
Mitochondria absent Mitochondria present
Cytoskeleton absent Cytoskeleton present
Ribosomes smaller Ribosomes larger
Vesicles present Vesicles present
Golgi apparatus absent Golgi apparatus present
Chloroplasts absent; chlorophyll scattered Chloroplasts present in plants
in the cytoplasm
Submicroscopic in size Flagella is present Microscopic in size, membrane-bound
and made up of only one fiber
Cell wall chemically complexed Cell wall is present in plants and fungi and chemically
simpler
Vacuoles absent Vacuoles present
Permeability of Nuclear membrane is not Permeability of Nuclear membrane is selective
present
Sexual reproduction is absent Sexual reproduction is present
Endocytosis and exocytosis are absent. Endocytosis and exocytosis occurred
It may have pili and fimbriae. Pili and fimbriae are absent
Transcription occurs in the cytoplasm Transcription occurs inside the nucleus.
Examples: Bacteria and Archaea Examples: Protists, Fungi, Plants, and Animals

THE NUCLEUS

The nucleus is a membrane bound organelle found in most eukaryotic cells. It is the largest
organelle of the eukaryotic cell, accounting for around 10% of its volume. It houses the genome,
and through gene expression, it co-ordinates the activities of the cell.

In general, a eukaryotic cell has only one nucleus. However, some eukaryotic cells are
enucleated cells (without a nucleus), for example, red blood cells (RBCs); whereas, some are
multinucleate (consists of two or more nuclei), for example, slime molds.
The nucleus is normally around 5-10 μm in diameter in many multicellular organisms, and the
largest organelle in the cell. The smallest nuclei are approximately 1 μm in diameter and are
found in yeast cells.

Structure of Nucleus

Nucleus Diagram

The structure of a nucleus encompasses the nuclear envelope, nucleoplasm, chromatin, and
nucleolus.

Nuclear envelope

The nucleus is surrounded by the nuclear envelope. This consists of both an inner and outer
membrane which run parallel to each other. The envelope is perforated by small gaps known as
the nuclear pores. These pores are around 100nm wide in true diameter, however due to the
presence of central regulatory proteins the true size of the gap is around 9nm.This small size
controls the passage of molecules into and out of the nucleus. Larger molecules such as larger
proteins and nucleic acid are unable to pass through these pores, and so the function of the
nuclear envelope is to selectively separate the contents of the nucleus from that of the cytoplasm.

Nuclear lamina

Mechanical support for the nucleus is provided by the nuclear lamina. This is a protein mesh,
which is found near the inner nuclear membrane. It is involved in most nuclear activities
including DNA replication, RNA transcription, nuclear and chromatin organization, cell cycle
regulation, etc
Nucleoplasm

Nucleoplasm is the gelatinous substance within the nuclear envelope. Also called karyoplasm,
this semi-aqueous material is similar to the cytoplasm and is composed mainly of water with
dissolved salts, enzymes, and organic molecules suspended within. The nucleolus and
chromosomes are surrounded by nucleoplasm, which functions to cushion and protect the
contents of the nucleus.

Nucleoplasm also supports the nucleus by helping to maintain its shape. Additionally,
nucleoplasm provides a medium by which materials, such as enzymes and nucleotides (DNA and
RNA subunits), can be transported throughout the nucleus. Substances are exchanged between
the cytoplasm and nucleoplasm through nuclear pores.

Chromatin

Chromatin describes DNA that is complexed with proteins. The primary protein components of
chromatin are histones, which are highly basic proteins that associate readily with DNA.
Histones combined with DNA form nucleosomes, which are the subunit of chromatin.
Specifically, a nucleosome describes a segment of DNA associated with 8 histone proteins. By
associating with histones, DNA is more compact and able to fit into the nucleus.

Chromatin can exist as either euchromatin or heterochromatin. Euchromatin is the form of

chromatin present during gene expression and has a characteristic ‘beads on a string’ appearance.
It is activated by acetylation. In contrast, heterochromatin is the ‘inactive’ form, and is densely
packed. On electron microscopy, euchromatin stains lighter than heterochromatin which reflects
their relative densities.

Nucleolus

Contained within the nucleus is a dense, membrane-less structure composed of RNA and
proteins called the nucleolus. Some of the eukaryotic organisms have a nucleus that contains up
to four nucleoli. The nucleolus contains nucleolar organizers, which are parts of chromosomes
with the genes for ribosome synthesis on them. The nucleolus helps to synthesize ribosomes by
transcribing and assembling ribosomal RNA subunits. These subunits join to form a ribosome
during protein synthesis. The nucleolus disappears when a cell undergoes division and is
reformed after the completion of cell division.

All these components work together in order for the nucleus to accomplish all of its functions.

Functions of Nucleus

The nucleus provides a site for genetic transcription that is segregated from the location of
translation in the cytoplasm, allowing levels of gene regulation that are not available to
prokaryotes. The main function of the cell nucleus is to control gene expression and mediate the
replication of DNA during the cell cycle.

It controls the hereditary characteristics of an organism.

The organelle is also responsible for protein synthesis, cell division, growth, and differentiation.

Storage of hereditary material, the genes in the form of long and thin DNA (deoxyribonucleic
acid) strands, referred to as chromatin.

Storage of proteins and RNA (ribonucleic acid) in the nucleolus.

The nucleus is a site for transcription in which messenger RNA (mRNA) are produced for
protein synthesis.

During the cell division, chromatins are arranged into chromosomes in the nucleus.

Production of ribosomes (protein factories) in the nucleolus.

Selective transportation of regulatory factors and energy molecules through nuclear pores.

Chromosome structure
Chromosomes are thread-like structures present in the nucleus. They are important because they
contain the basic genetic material DNA. These are present inside the nucleus of plants as well as
animal cells. Chromosomes were first discovered by Strasburger in 1815 and the term
‘chromosome’ was first used by Waldeyer in 1888. Human beings have 46 chromosomes in their
body. These are arranged into 23 pairs.

Points to note
  In the nucleus of each cell, the DNA molecule is packaged into thread-like structures
called chromosomes.
 Each chromosome is made up of DNA tightly coiled many times around proteins called
histones that support its structure.
 Chromosomes were first described by Strasburger (1815), and the term ‘chromosome’
was first used by Waldeyer in 1888.
 They appear as rod shaped dark stained bodies during the metaphase stage of mitosis
when cells are stained with a suitable basic dye and viewed under a light microscope.

Chromosome structure

 In eukaryotes the chromosomes are multiple large, linear and are present in the nucleus of
the cell.
 Each chromosome typically has one centromere and one or two arms that project from
the centromere.
 Structurally, each chromosome is differentiated into three parts—
1. Pellicle
2. Matrix
3. Chromonemata
Pellicle
 It is the outer envelope around the substance of chromosome.
 It is very thin and is formed of achromatic substances.
Matrix
 It is the ground substance of chromosome which contains the chromonemata.
  It is also formed of non-genic materials.
Chromonemata
 Embedded in the matrix of each chromosome are two identical, spirally coiled threads, the
chromonemata.
 The two chromonemata are also tightly coiled together that they appear as single thread of
about 800A thickness.
 Each chromonemata consists of about 8 microfibrils, each of which is formed of a double
helix of DNA.
Centromere
 A small structure in the chromonema, marked by a constriction which is recognized as
permanent structure in the chromosome is termed as the centromere.
 At this point the two chromonemata are joined together.
 It is known as centromere or kinetochore or primary constriction.
 It divides the chromosome into two sections, or “arms.” The short arm of the
chromosome is labeled the “p arm.” The long arm of the chromosome is labeled the “q
arm.”
 Its position is constant for a given type of chromosome and forms a feature of
identification.
 In thin electron microscopic sections, the kinetochore shows a trilaminar structure, i.e., a
10 nm thick dense outer protein aqueous layer, a middle layer of low density and a dense
inner layer tightly bound to the centromere.
 The chromosomes are attached to spindle fibres at this region during cell division.
Secondary constriction or Nucleolar organizer
 The chromosome besides having the primary constriction or the centromere possesses
secondary constriction at any point of the chromosome.
 Constant in their position and extent, these constrictions are useful in identifying
particular chromosomes in a set.
 The chromosome region distal to the secondary constriction i.e., the region between the
secondary constriction and the nearest telomere is known as satellite.
 Therefore, chromosomes having secondary constrictions are called satellite chromosomes
or sat-chromosomes.
  Nucleolus is always associated with the secondary constriction of sat-chromosomes.
Therefore, secondary constrictions are also called nucleolus organizer region (NOR) and
sat-chromosomes are often referred to as nucleolus organizer chromosomes.
Telomere
 These are specialized ends of a chromosome which exhibits physiological differentiation
and polarity.
 Each extremity of the chromosome due to its polarity prevents other chromosomal seg-
ments to be fused with it. The chromosomal ends are known as the telomeres.
 If a chromosome breaks, the broken ends can fuse with each other due to lack of
telomere.
Types of Chromosomes
A) Autosomes and Sex chromosomes
Human chromosomes are of two types autosomes and sex chromosomes.
Genetic traits that are linked to the sex of the person are passed on through the sex chromosomes.
The rest of the genetic information is present in the autosomes.
Humans have 23 pairs of chromosomes in their cells, of which 22 pairs are autosomes and one
pair of sex chromosomes, making a total of 46 chromosomes in each cell.
B) On the basis of number of centromeres
Monocentric with one centromere.
Dicentric with two centromeres.
Polycentric with more than two centromeres
Acentric without centromere. Such chromosomes represent freshly broken segments of
chromosomes which do not survive for long.
Diffused or non-located with indistinct centromere diffused throughout the length of
chromosome.
C) On the basis of location of centromere
 1. Telocentric are rod-shaped chromosomes with centromere occupying the terminal
position, so that the chromosome has just one arm.
2. Acrocentric are also rod-shaped chromosomes with centromere occupying a sub-
terminal position. One arm is very long and the other is very short.
3. Sub-metacentric chromosomes are with centromere slightly away from the mid-point so
that the two arms are unequal.
4. Metacentric are V-shaped chromosomes in which centromere lies in the middle of chro-
mosome so that the two arms are almost equal.

SPECIAL CHROMOSOMES
Polytene Chromsomes or Salivary Gland Chromosomes
The giant chromosomes were first observed in the cells of salivary glands, gut, trachea and other
body parts of dipteran insects by E.G. Balbiani in 1881. The name polytene was assigned to
these chromosomes by Kollar due to the occurrence of many chromonemata (DNA) in them. The
polytene chromosomes are much larger than the normal somatic chromosomes.

The giant chromosomes consist of a bundle of chromonemal fibrils which arise by a series of
about 10 consecutive duplications of the initial chromonemata that increase the DNA content
about 1,000 times the DNA content of somatic cells. Because of the multi-stranded condition,
these chromosomes are called polytene chromosomes.

Polyteney of the giant chromosomes happens by replication of the chromosomal DNA several
times without nuclear division (endomitosis) and the resulting daughter chromatids do no
separate but remain aligned side by side. During endomitosis, the nuclear envelope does not
rupture and no spindle formation takes place. The polytene chromosomes are visible during
interphase and prophase of mitosis. A series of dark transverse bands alternates with clear zones
of inter bands. Such individual bands can be correlated with particular genes. About 85% of the
DNA in polytene chromosomes is in bands and 15% is in inter bands. The cross-banding pattern
of each polytene chromosome is a constant characteristic within a species and helps in
chromosome mapping during cytogenetic studies. These chromosomes are not inert cellular
objects but dynamic structures in which certain regions become “puffed out” due to active DNA
transcription at particular stages of development. these chromosome puffs are also termed
Balbiani rings. Puffs may appear and disappear depending on the production of specific proteins
which needs to be secreted in large amounts in the larval saliva. Another peculiarity of the
polytene chromosomes is that the paternal and maternal chromosomes remain associated side by
side and the phenomenon is termed somatic pairing.

Lampbrush chromosome
Lampbrush chromosomes were first observed by Flemming in 1882 in sections of Salamander
oocytes and later described by Ruckert in the year 1892. They appeared like brushes used for
cleaning lamps, hence the name lampbrush chromosome. They are transitory structures and can
be observed during the diplotene stage of prophase I in meiosis in the oocytes of all animal
species both vertebrates and invertebrates except in mammals. They have been described in
Sepia (Mollusca), Echinaster (Echinodermata) and in several species of insects, shark,
amphibians, reptiles and birds. Lampbrush chromosomes have also been found in spermatocytes
of several species, giant nucleus of Acetabularia and even in plants. Generally, they are smaller
in invertebrates than vertebrates. They are observed in oocytes because oocytes are high in DNA
content. Lampbrush chromosomes are functional for studying chromosome organization and
genome function during meiotic prophase. Additionally, lampbrush chromosomes are widely
used for construction of detail cytological maps of individual chromosomes. They are of
exceptionally large sizes and present in bivalent form. They are formed due to the active
synthesis of mRNA molecules for future use by the egg cells, when no synthesis of mRNA
molecule is possible during the mitotic cell division. Lampbrush chromosomes are clearly visible
in the light microscope and they are organized into a series of chromomeres with large chromatin
symmetrical loops extending laterally. Each loop appears at a constant position in the
chromosome (10,000 loops per chromosome set or haploid set). Each loop has an axis made up
of DNA unfolded from the chromosome and is transcriptionally highly active. Wherein several
transcription units with polarized ribonucleoprotein matrix (RNP-matrix) coats the DNA axis of
the loop. The majority of the DNA, however, is not in loops but remains highly condensed in the
chromomeres on the axis and lacks expression of genes.

B-chromosomes

Many plant (maize, etc.) and animal (such as insects and small mammals) species, besides
having autosomes (A-chromosomes) and sex-chromosomes possess a special category of
chromosomes called B-chromosomes without obvious genetic function. These B-chromosomes
(also called supernumerary chromosomes, accessory chromosomes, accessory fragments, etc.)
usually have a normal structure, are somewhat smaller than the autosomes and can be
predominantly, heterochromatic (many insects, maize, etc.) or pre-dominantly euchromatic (rye).
In maize, their number per cell can vary from 0 to 30 and they adversely affect, development and
fertility only when they occur in large amount. In animals, the B-chromosomes disappear from
the non-reproductive (somatic) tissue and are maintained only in the cell-lines that lead to the
reproductive organs. B-chromosomes have negative consequences for the organism, as they have
deleterious effect because of abnormal crossing over during the meiosis of animals and abnormal
nucleus divisions of the gametophyte plants. In animals, B-chromosomes occur more frequently
in females and the basis is non-disjunction. The non- disjunction of B-chromosomes of rye plant
is found to be caused due to the presence of a heterochromatic knob at the end of long arm of B-
chromosome. The origin of the B-chromosomes is uncertain. In some animals they may be
derivatives of sex chromosomes, but this is not the rule. They generally do not show any pairing
affinity with the A-chromosomes.
 B-chromosomes
CELL CYCLE
The cell cycle is the ordered sequence of events that occur in a cell in preparation for cell
division. The cell cycle is a four-stage process in which the cell increases in size (gap 1, or G1,
stage), copies its DNA (synthesis, or S, stage), prepares to divide (gap 2, or G2, stage), and
divides (mitosis, or M, stage). The stages G1, S, and G2 make up interphase, which accounts for
the span between cell divisions. On the basis of the stimulatory and inhibitory messages a cell
receives, it “decides” whether it should enter the cell cycle and divide.

The length of the cell cycle is highly variable, even within the cells of a single organism. In
humans, the frequency of cell turnover ranges from a few hours in early embryonic development,
to an average of two to five days for epithelial cells, and to an entire human lifetime spent in G 0
by specialized cells, such as cortical neurons or cardiac muscle cells. There is also variation in
the time that a cell spends in each phase of the cell cycle. When fast-dividing mammalian cells
are grown in culture (outside the body under optimal growing conditions), the length of the cycle
is about 24 hours. In rapidly dividing human cells with a 24-hour cell cycle, the G 1 phase lasts
approximately nine hours, the S phase lasts 10 hours, the G 2 phase lasts about four and one-half
hours, and the M phase lasts approximately half hour. In early embryos of fruit flies, the cell
cycle is completed in about eight minutes. The timing of events in the cell cycle is controlled by
mechanisms that are both internal and external to the cell.

Regulation of the Cell Cycle by External Events

Both the initiation and inhibition of cell division are triggered by events external to the cell when
it is about to begin the replication process. An event may be as simple as the death of a nearby
cell or as sweeping as the release of growth-promoting hormones, such as human growth
hormone (HGH). A lack of HGH can inhibit cell division, resulting in dwarfism, whereas too
much HGH can result in gigantism. Crowding of cells can also inhibit cell division. Another
factor that can initiate cell division is the size of the cell; as a cell grows, it becomes inefficient
due to its decreasing surface-to-volume ratio. The solution to this problem is to divide.

Whatever the source of the message, the cell receives the signal, and a series of events within the
cell allows it to proceed into interphase. Moving forward from this initiation point, every
parameter required during each cell cycle phase must be met or the cycle cannot progress.

Regulation at Internal Checkpoints

It is essential that the daughter cells produced be exact duplicates of the parent cell. Mistakes in
the duplication or distribution of the chromosomes lead to mutations that may be passed forward
to every new cell produced from an abnormal cell. To prevent a compromised cell from
continuing to divide, there are internal control mechanisms that operate at three main cell cycle
checkpoints. A checkpoint is one of several points in the eukaryotic cell cycle at which the
progression of a cell to the next stage in the cycle can be halted until conditions are favorable.
These checkpoints occur near the end of G1, at the G2/M transition, and during metaphase

The G1 Checkpoint

The G1 checkpoint determines whether all conditions are favorable for cell division to proceed.
The G1 checkpoint, also called the restriction point (in yeast), is a point at which the cell
irreversibly commits to the cell division process. External influences, such as growth factors,
play a large role in carrying the cell past the G 1 checkpoint. In addition to adequate reserves and
cell size, there is a check for genomic DNA damage at the G 1 checkpoint. A cell that does not
meet all the requirements will not be allowed to progress into the S phase. The cell can halt the
cycle and attempt to remedy the problematic condition, or the cell can advance into G 0 and await
further signals when conditions improve.

The G2 Checkpoint

The G2 checkpoint bars entry into the mitotic phase if certain conditions are not met. As at the G 1
checkpoint, cell size and protein reserves are assessed. However, the most important role of the
G2 checkpoint is to ensure that all of the chromosomes have been replicated and that the
replicated DNA is not damaged. If the checkpoint mechanisms detect problems with the DNA,
the cell cycle is halted, and the cell attempts to either complete DNA replication or repair the
damaged DNA.

The M Checkpoint

The M checkpoint occurs near the end of the metaphase stage of karyokinesis. The M checkpoint
is also known as the spindle checkpoint, because it determines whether all the sister chromatids
are correctly attached to the spindle microtubules. Because the separation of the sister chromatids
during anaphase is an irreversible step, the cycle will not proceed until the kinetochores of each
pair of sister chromatids are firmly anchored to at least two spindle fibers arising from opposite
poles of the cell.

CELL CYCLE – DNA REPLICATION

Replicating DNA is fragile, and can break during the duplication process. In fact, broken
chromosomes are often the source of DNA rearrangements and can change the genetic program
of a cell. These changes can trigger a growth advantage in a single cell and when that cell
continues to divide, tumors arise. Fortunately, cells have defense mechanisms to shield the body
from these damaging events.
In the eukaryotic cell cycle, chromosome duplication occurs during "S phase" (the phase of DNA
synthesis) and chromosome segregation occurs during "M phase" (the mitosis phase). During S
phase, any problems with DNA replication trigger a ‘'checkpoint". The S phase checkpoint
operates like a surveillance camera.

Double stranded DNA is intrinsically more stable than single stranded DNA, although the latter
can be stabilized and protected by single-strand DNA binding proteins. Researchers have
recently discovered that, in eukaryotes, the replication protein A (RPA) is a form of red flag in
the cell: when RPA is coating long strands of ssDNA, this signals a checkpoint. This concept
underscores an important feature: presence of ssDNA signals that "something is wrong" and this
also holds true for other phases of the cell cycle. In other words, whether ssDNA is created
during replication, or outside of S phase, it will always trigger the checkpoint surveillance
system. Interestingly, this phenomenon is also present at unprotected telomeres (chromosome
ends) that contain ssDNA.

RPA-coated ssDNA attracts a specific protein with a complicated name: the ataxia telangiectasia
mutated and Rad3 related kinase, also known as ATR. ATR associates with RPA and activates
its intrinsic kinase activity. This starts a signal that temporarily halts S phase progression.
Therefore, ATR is also known as the S phase "checkpoint kinase."

ATR kinase acts in several ways to keep the replication process intact. There is evidence that
ATR also stabilizes replication forks that contain ssDNA. How this happens remains largely
unclear.

There are other circumstances that cause replication to go awry. One is that the DNA template
somehow becomes defective during replication, and causes the polymerase to pause. For
example, a DNA base can be chemically modified or spontaneously altered. This generates a
lesion — an area that is a roadblock for DNA polymerases and DNA primase. Therefore, DNA
lesions cause regions of DNA to remain single-stranded (uncopied).

Scientists use the term "stalled forks" for areas of replication forks where DNA polymerization is
halted. Stalled forks activate ATR, which in turn phospohorylates its downstream target, the
checkpoint kinase 1 (Chk1). Little is known about the phosphorylation targets that lie further
downstream of Chk1, but when scientists observe Chk1 phosphorylation in cells, they conclude
that cells are actively trying to protect replication forks with DNA lesions.

MITOSIS
Mitosis is a process of cell duplication or reproduction where a single cell divides into two
identical daughter cells (cell division).
 During mitosis one cell divides once to form two identical cells.
 The major purpose of mitosis is for growth and to replace worn out cells.
 If not corrected in time, mistakes made during mitosis can result in changes in the DNA
that can potentially lead to genetic disorders.
Mitosis is divided into four phases:
1. Prophase:
 The chromosomes condense into X-shaped structures that can be easily seen under a
microscope.
 Each chromosome is composed of two sister chromatids, containing identical genetic
information.
 The chromosomes pair up so that both copies of chromosome 1 are together, both copies
of chromosome 2 are together, and so on.
 At the end of prophase, the membrane around the nucleus in the cell dissolves away
releasing the chromosomes.
 The mitotic spindle, consisting of the microtubules and other proteins, extends across the
cell between the centrioles as they move to opposite poles of the cell.
2. Metaphase:
 The chromosomes line up neatly end-to-end along the center (equator) of the cell.
 The centrioles are now at opposite poles of the cell with the mitotic spindle fibres
extending from them.
 The mitotic spindle fibres attach to each of the sister chromatids.
3. Anaphase:
 The sister chromatids are then pulled apart by the mitotic spindle which pulls one
chromatid to one pole and the other chromatid to the opposite pole.
4. Telophase:
 At each pole of the cell a full set of chromosomes gather together.
  A membrane forms around each set of chromosomes to create two new nuclei.
 The single cell then pinches in the middle to form two separate daughter cells each
containing a full set of chromosomes within a nucleus. This process is known as
cytokinesis.

MEIOSIS
Meiosis is a special type of cell division of germ cells in sexually-reproducing organisms used to
produce the gametes, such as sperm or egg cells. It involves two rounds of division that
ultimately result in four cells with only one copy of each paternal and maternal chromosome
(haploid). Additionally, prior to the division, genetic material from the paternal and maternal
copies of each chromosome is crossed over, creating new combinations of code on each
chromosome. Later on, during fertilization, the haploid cells produced by meiosis from a male
and female will fuse to create a cell with two copies of each chromosome again, the zygote. The
process of meiosis begins with a cell with double the normal amount of DNA, and ends up with
4 non-identical haploid daughter gametes, after two divisions.
Before meiosis begins, during S phase of the cell cycle, the DNA of each chromosome is
replicated so that it consists of two identical sister chromatids, which remain held together
through sister chromatid cohesion. This S-phase can be referred to as "premeiotic S-phase" or
"meiotic S-phase". Immediately following DNA replication, meiotic cells enter a prolonged G2-
like stage known as meiotic prophase. During this time, homologous chromosomes pair with
each other and undergo genetic recombination, a programmed process in which DNA may be cut
and then repaired, which allows them to exchange some of their genetic information. A subset of
recombination events results in crossovers, which create physical links known as chiasmata
(singular: chiasma, for the Greek letter Chi (X)) between the homologous chromosomes. In most
organisms, these links can help direct each pair of homologous chromosomes to segregate away
from each other during Meiosis I, resulting in two haploid cells that have half the number of
chromosomes as the parent cell.

During meiosis II, the cohesion between sister chromatids is released and they segregate from
one another, as during mitosis. In some cases, all four of the meiotic products form gametes such
as sperm, spores or pollen. In female animals, three of the four meiotic products are typically
eliminated by extrusion into polar bodies, and only one cell develops to produce an ovum.
Because the number of chromosomes is halved during meiosis, gametes can fuse (i.e.
fertilization) to form a diploid zygote that contains two copies of each chromosome, one from
each parent. Thus, alternating cycles of meiosis and fertilization enable sexual reproduction, with
successive generations maintaining the same number of chromosomes. For example, diploid
human cells contain 23 pairs of chromosomes including 1 pair of sex chromosomes (46 total),
half of maternal origin and half of paternal origin. Meiosis produces haploid gametes (ova or
sperm) that contain one set of 23 chromosomes. When two gametes (an egg and a sperm) fuse,
the resulting zygote is once again diploid, with the mother and father each contributing 23
chromosomes. This same pattern, but not the same number of chromosomes, occurs in all
organisms that utilize meiosis.

There are six stages within each of the divisions, namely prophase, prometaphase, metaphase,
anaphase, telophase and cytokinesis.

Meiosis I
In meiosis I, homologous chromosomes are separated into two cells such that there is one
chromosome (consisting of two chromatids) per chromosome pair in each daughter cell.
Prophase I
Prior to prophase, chromosomes replicate to form sister chromatids. There are initially four
chromatids (c) and two chromosomes (n) for each of the 23 chromosome pairs (4c, 2n). The
nuclear envelope disintegrates and the chromosomes begin to condense. Spindle fibres appear
which will be important for successful division of the chromosomes.
To further increase the genetic diversity, homologous chromosomes exchange parts of
themselves such that one chromosome contains both maternal and paternal DNA. This process is
known as crossing over, and the points at which this occurs on a chromosome are referred to as
chiasmata.
Prophase I is by far the longest phase of meiosis (lasting 13 out of 14 days in mice). During
prophase I, homologous maternal and paternal chromosomes pair, synapse, and exchange genetic
information (by homologous recombination), forming at least one crossover per chromosome.
These crossovers become visible as chiasmata (plural; singular chiasma). This process facilitates
stable pairing between homologous chromosomes and hence enables accurate segregation of the
chromosomes at the first meiotic division. The paired and replicated chromosomes are called
bivalents (two chromosomes) or tetrads (four chromatids), with one chromosome coming from
each parent. Prophase I is divided into a series of substages which are named according to the
appearance of chromosomes.
Leptotene
The first stage of prophase I is the leptotene stage, also known as leptonema, from Greek words
meaning "thin threads”. In this stage of prophase I, individual chromosomes—each consisting of
two replicated sister chromatids—become "individualized" to form visible strands within the
nucleus. The chromosomes each form a linear array of loops mediated by cohesion, and the
lateral elements of the synaptonemal complex assemble forming an "axial element" from which
the loops emanate. Recombination is initiated in this stage by the enzyme SPO11 which creates
programmed double strand breaks.
Zygotene
Leptotene is followed by the zygotene stage, also known as zygonema, from Greek words
meaning "paired threads". In this stage the homologous chromosomes become much more
closely (~100 nm) and stably paired (a process called synapsis) mediated by the installation of
the transverse and central elements of the synaptonemal complex. Synapsis is thought to occur in
a zipper-like fashion starting from a recombination nodule. The paired chromosomes are called
bivalent or tetrad chromosomes.
Pachytene
The pachytene stage, also known as pachynema, from Greek words meaning "thick threads" is
the stage at which all autosomal chromosomes have synapsed. In this stage homologous
recombination, including chromosomal crossover (crossing over), is completed through the
repair of the double strand breaks formed in leptotene.
Diplotene
During the diplotene stage, also known as diplonema, from Greek words meaning "two threads",
the synaptonemal complex disassembles and homologous chromosomes separate from one
another a little. However, the homologous chromosomes of each bivalent remain tightly bound at
chiasmata, the regions where crossing-over occurred. The chiasmata remain on the chromosomes
until they are severed at the transition to anaphase I to allow homologous chromosomes to move
to opposite poles of the cell.
Diakinesis
Chromosomes condense further during the diakinesis stage, from Greek words meaning "moving
through". This is the first point in meiosis where the four parts of the tetrads are actually visible.
Sites of crossing over entangle together, effectively overlapping, making chiasmata clearly
visible. Other than this observation, the rest of the stage closely resembles prometaphase of
mitosis; the nucleoli disappear, the nuclear membrane disintegrates into vesicles, and the meiotic
spindle begins to form.
Prometaphase I
Now the spindle fibres attach to the chromosomes at a point along the chromosomes called
centromeres. While this is happening, the chromosomes continue to condense.
Metaphase I
Next, maternal and paternal versions of the same chromosome align along the equator of the cell.
These are the homologous chromosomes. A process called independent assortment occurs –
this is when maternal and paternal chromosomes line up randomly align themselves on either
side of the equator. This in turn determines to which gamete chromosomes are allocated to,
which leads to genetic diversity among offspring.

Anaphase I
Here each of the homologous chromosomes get pulled towards opposite poles of the cell as the
spindle fibres retract to divide the DNA between the two cells which will be formed.
Telophase I and Cytokinesis I
During telophase I, the nuclear envelope reforms and spindle fibres disappear. In Cytokinesis I,
the cytoplasm and cell divides resulting in two cells that are technically haploid – there is one
chromosome and two chromatids for each chromosome (2c, n).

Meiosis II
Prophase II and Prometaphase II
These stages are identical to their counterparts in meiosis I.
Metaphase II
Now chromosomes line up in single file along the equator of the cell. This is in contrast to
Metaphase I where chromosomes lined up in homologous pairs.
Anaphase II
Next, sister chromatids are pulled to opposite poles of the equator.

Telophase II
This is the same as Telophase I.
Cytokinesis II
Again, the cytoplasm and cell divides producing 2 non-identical haploid daughter cells, but as
this is happening in both cells produced by meiosis I, the net product is 4 non-identical haploid
daughter cells, each comprising one chromosome consisting of one chromatid (1c, 1n). These are
gametes.

Meiosis occurs in all animals and plants. The end result, the production of gametes with half the
number of chromosomes as the parent cell, is the same, but the detailed process is different. In
animals, meiosis produces gametes directly. In land plants and some algae, there is an alternation
of generations such that meiosis in the diploid sporophyte generation produces haploid spores.
These spores multiply by mitosis, developing into the haploid gametophyte generation, which
then gives rise to gametes directly (i.e. without further meiosis). In both animals and plants, the
final stage is for the gametes to fuse, restoring the original number of chromosomes.