MEIOSIS

Meiosis is a specialized type of cell division which

reduces the chromosome number by half. This process
occurs in all sexually reproducing single-celled and
multi-celled eukaryotes, including animals, plants, and
fungi.Errors in meiosis resulting in aneuploidy are the
leading known cause of miscarriage and the most
frequent genetic cause of developmental disabilities.
Meiosis is a process where a single cell divides twice
to produce four cells containing half the original
amount of genetic information. These cells are our sex
cells sperm in males, eggs in females.
Meiosis is the process whereby chromosomes are
copied, paired up and separated to create eggs or
sperm.
In meiosis, the chromosomes duplicate (during
interphase) and homologous chromosomes exchange
genetic information (chromosomal crossover) before a
first division, called meiosis I. The daughter cells
divide again in meiosis II, splitting up sister
chromatids to form haploid gametes. Male and female
gametes fuse during fertilization, creating a diploid
cell with a complete set of paired chromosomes.
Meiosis can be divided into nine stages. These are
divided between the first time the cell divides
(meiosis I) and the second time it divides (meiosis II):

MEIOSIS 1

Interphase
The DNA in the cell is copied resulting in two identical
full sets of chromosomes.
Outside of the nucleus, are two centrosomes, each
containing a pair of centrioles, these structures are
critical for the process of cell division?
During interphase, microtubules extend from these
centrosomes.

Prophase 1
The copied 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.
The pairs of chromosomes may then exchange bits of
DNA in a process called recombination or crossing
over.
At the end of Prophase, I the membrane around the
nucleus in the cell dissolves away, releasing the
chromosomes.
The meiotic spindle, consisting of microtubules and
other proteins, extends across the cell between the
centrioles.

Metaphase I:

The chromosome pairs line up next to each other

along the centre (equator) of the cell.
The centrioles are now at opposites poles of the cell
with the meiotic spindles extending from them.
The meiotic spindle fibres attach to one chromosome
of each pair.

Anaphase I:

The pair of chromosomes are then pulled apart by the

meiotic spindle, which pulls one chromosome to one
pole of the cell and the other chromosome to the
opposite pole.
In meiosis I the sister chromatids stay together. This is
different to what happens in mitosis and meiosis II.

Telophase I and cytokinesis:

The chromosomes complete their move to the

opposite poles of the cell.
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 II
Prophase II:

Now there are two daughter cells, each with 23

chromosomes (23 pairs of Chromatids)

In each of the two daughter cells the chromosomes

condense again into visible X-shaped structures that can be
easily seen under a microscope.

The membrane around the nucleus in each daughter

cell dissolves away releasing the chromosomes.

The centrioles duplicate.

The meiotic spindle forms again.

Metaphase II:

In each of the two daughter cells the chromosomes

(pair of sister chromatids) line up end-to-end along the
equator of the cell.

The centrioles are now at opposites poles in each of

the daughter cells.

Meiotic spindle fibres at each pole of the cell attach to

each of the sister chromatids.
Anaphase II:

The sister chromatids are then pulled to opposite

poles due to the action of the meiotic spindle.

The separated chromatids are now individual

chromosomes.
Anaphase II:

The sister chromatids are then pulled to opposite

poles due to the action of the meiotic spindle.

The separated chromatids are now individual

chromosomes.
Telophase II and cytokinesis:

The chromosomes complete their move to the

opposite poles of the cell.

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 cell nuclei.

This is the last phase of meiosis, however cell division

is not complete without another round of cytokinesis.

Once cytokinesis is complete there are four

granddaughter cells, each with half a set of chromosomes
(haploid):
o
in males, these four cells are all sperm cells
o
in females, one of the cells is an egg cell while the
other three are polar bodies (small cells that do not develop
into eggs).
The importance of meiosis
Proper segregation of chromosomes during meiosis I and II is
essential to generate healthy sperm and egg cells, and by
extension, healthy embryos. Failed chromosomal
segregation is called nondisjunction, and can result in
gametes with missing chromosomes or extra chromosomes,
according to the authors of "Molecular Biology of the Cell."

Mitosis
WHAT IS MITOSIS?
Mitosis is a part of the cell cycle in which
chromosomes in a cell nucleus are separated into two
identical sets of chromosomes, each in its own nucleus.
Mitosis (division of the nucleus) is often followed
by cytokinesis, which divides the cytoplasm,

organelles and cell membrane into two new cells containing

roughly equal shares of these cellular components.
Mitosis and cytokinesis together define
the mitotic (M) phase of an animal cell cyclethe division of
the mother cell into two daughter cells, genetically identical
to each other and to their parent cell.
Mitosis is a part of the cell cycle in which
chromosomes in a cell nucleus are separated into two
identical sets of chromosomes, each in its own nucleus.
Mitosis (division of the nucleus) is often followed
by cytokinesis, which divides the cytoplasm,
organelles and cell membrane into two new cells containing
roughly equal shares of these cellular components.
Mitosis and cytokinesis together define
the mitotic (M) phase of an animal cell cyclethe division of
the mother cell into two daughter cells, genetically identical
to each other and to their parent cell.
Discovery
German zoologist Otto Btschli might have claimed the
discovery of the process presently known as "mitosis", a
term coined by Walther Flemming in 1882.
Mitosis was discovered in frog, rabbit, and
cat cornea cells in 1873 and described for the first time by
the Polish historologist, Wacaw Mayzel in 1875. The term is
derived from the Greek word () mitos "warp thread".

1st Prophase: The two round objects above the nucleus are
the centrosomes. The chromatin is condensing into
chromosomes.
2nd Prometaphase: The nuclear envelope disintegrates, and
microtubules have invaded the nuclear space. These
microtubules can attach to kinetochores or they can interact
with opposing microtubules.
3rd Metaphase: The chromosomes align at the metaphase
plate.
4th Anaphase: The chromosomes split and the kinetochore
microtubules shorten.
5th Telophase: The decondensing chromosomes are
surrounded by newly formed nuclear envelopes. Cytokinesis
has already begun; the pinched area is known as
the cleavage furrow.
The Cell Cycle
Mitosis is nuclear division plus cytokinesis, and produces
two identical daughter cells during prophase, prometaphase,
metaphase, anaphase, and telophase.

1st
Interphase

The process of mitosis is divided into stages

corresponding to the completion of one set of activities and
the start of the next.
Phases of Mitosis

2nd

The mitotic phase is a relatively short period

of the cell cycle. It alternates with the much
longer interphase, where the cell prepares
itself for the process of cell division.
Interphase is divided into three
phases: G1 (first gap), S (synthesis), and G2
(second gap). Thus, a cell grows (G1),
continues to grow as it duplicates its
chromosomes (S), grows more and prepares
for mitosis (G2), and finally divides (M) before
restarting the cycle.
Chromatin in the nucleus begins to condense
and becomes visible in the light microscope as

Prophase

chromosomes. The nucleolus disappears.

Centrioles begin moving to opposite ends of
the cell and fibers extend from the
centromeres.

The nuclear membrane dissolves, marking the

beginning of prometaphase. Proteins attach to
Prometaphas the centromeres creating the kinetochores.
Microtubules attach at the kinetochores and
e
the chromosomes begin moving.
3rd

4th
Metaphase

5th
Anaphase

6th
Telophase

7th
Cytokinesis

Spindle fibers align the chromosomes along

the middle of the cell nucleus. This line is
referred to as the metaphase plate. This
organization helps to ensure that in the next
phase, when the chromosomes are separated,
each new nucleus will receive one copy of
each chromosome.
The paired chromosomes separate at the
kinetochores and move to opposite sides of
the cell. Motion results from a combination of
kinetochore movement along the spindle
microtubules and through the physical
interaction of polar microtubules.
Chromatids arrive at opposite poles of cell,
and new membranes form around the
daughter nuclei. The chromosomes disperse
and are no longer visible under the light
microscope. The spindle fibers disperse, and
cytokinesis or the partitioning of the cell may
also begin during this stage.
In animal cells, cytokinesis results when a fiber
ring composed of a protein called actin around
the center of the cell contracts pinching the
cell into two daughter cells, each with one

nucleus. In plant cells, the rigid wall requires

that a cell plate be synthesized between the
two daughter cells.
During mitosis, the chromosomes, which have already
duplicated, condense and attach to fibers that pull one copy
of each chromosome to opposite sides of the cell. The result
is two genetically identical daughter nuclei. The cell may
then divide by cytokinesis to produce two daughter cells.
Errors and variations of mitosis
Producing three or more daughter cells instead of normal
two is a mitotic error called tripolar mitosis or multipolar
mitosis (direct cell triplication / multiplication). Other errors
during mitosis can induce apoptosis (programmed cell
death) or cause mutations. Certain types of cancer can arise
from such mutations.
In nondisjunction, sister chromatids fail to separate during
anaphase. One daughter cell receives both sister chromatids
from the non-disjoining chromosome and the other cell
receives none. As a result, the former cell gets three copies
of the chromosome, a condition known as trisomy, and the
latter will have only one copy, a condition known as
monosomy. On occasion, when cells experience
nondisjunction, they fail to complete cytokinesis and retain
both nuclei in one cell, resulting in binucleated cells.

DNA REPLICATION
What is dna?

DNA, or deoxyribonucleic acid, is the hereditary

material in humans and almost all other organisms.
DNA is a molecule that carries most of the genetic
instructions.

Enzymes

Often end in -ase

Have the ability to speed up reaction and build up or
break down the items that they act on

Kinds of enzymes
Helicase

The unzipping enzyme

Also known as helix destabilizing enzyme
Unwinds the DNA double helix at the Replication Fork

Dna polymerase

The builder
Replicates DNA molecules to build DNA strands.
Builds a new duplex DNA strand by adding nucleotides
in the 5' to 3' direction.
Also performs proof-reading and error correction.
Needs a primer which the primase creates for it to
start building.

Single-Strand Binding (SSB) Proteins

Enzyme that seals up the fragments into one long

continuous strand.

Primase

Primase is an enzyme that synthesizes short RNA

sequences called primers. These primers serve as a
starting point for DNA synthesis.

DNA Ligase

Enzyme that seals up the fragments into one long

continuous strand.

How DNA Replication Works

Replication Process
Initiation

For a cell to divide, it must first replicate its DNA.

This process is initiated at particular points in the
DNA, known as "origins", which are targeted by
initiator proteins.
Once the origin has been located, these initiators
recruit other proteins and form the pre-replication
complex, which unzips the double-stranded DNA.

Termination

Requires that the progress of the DNA replication fork

must stop or be blocked.
Occurs when the two replication forks meet each
other on the opposite end of the parental
chromosome.

Requires that the progress of the DNA replication fork

must stop or be blocked.
Occurs when the two replication forks meet each
other on the opposite end of the parental
chromosome.
Involves the interaction between two components:
1. a termination site sequence in the DNA, and
2. a protein which binds to this sequence to physically
stop DNA replication.

DNA TRANSCRIPTION
What is Transcription?
It is the first step of gene expression, in which a
particular segment of DNA is copied into RNA(mRNA)
by the enzyme RNA polymerase.

3 Main types of RNA

(mRNA) Messenger RNA

(rRNA) Ribosomal RNA
(tRNA) Transfer RNA

Transcription involves four stages.

BINDING

RNA polymerase begins synthesizing RNA from the

template strands of the DNA as the DNA helix unwinds
farther.

ELONGATION

Termination

INITIATION

Binding of the RNA polymerase to a specific sequence,

called a promoter. The DNA helix unwinds in this
region.

As the RNA polymerase moves along the DNA strands

in the 3 to 5 direction, the DNA helix unwinds and
the two strands separate.
The RNA elongates by the addition of ribonucleotides
to the 3 end of the newly synthesized RNA.

TERMINATION

After the end of the gene is reached, termination

occurs:
The RNA polymerase disengages the DNA, and the
new RNA released.
At some termination sites in bacteria, rho proteins are
involved.
At other sites, termination is signaled by the formation
of a stem loop structure at the end of the new RNA
that forces the RNA polymerase to fall off the DNA
template.

KEY FACTORS INVOLVED IN TRANSCRIPTION

TATA BOX

Is a DNA sequence that indicates where a genetic

sequence can be read and decoded.
It is a type of promoter sequence, which specifies to
other molecules where transcription begins

ADENOSINE TRIPHOSPHATE (ATP)

Is a nucleoside triphosphate used in cells as a

coenzyme often called the molecular unit of
currency of intracellular energy transfer.
It transports chemical energy within cells for
metabolism.

DEOXYRIBONUCLEIC ACID (DNA)

A molecule that carries most of the genetic instruction

use in the development, functioning and reproduction
of all known living organisms and many viruses.

HISTORY OF GENETIC ENGINEERING

-

TRANSCRIPTION FACTOR

A protein that binds to specific DNA sequence,

thereby controlling the rate of transcription of genetic
information from DNA to messenger RNA.
It contains one or more DNA-binding domains (DBDs),
which attach to specific sequences of DNA adjacent to
the genes that they regulate.

RNA POLYMERASE

is an enzyme that produces primary transcript RNA

HISTORY
A molecule that allows the genetic material to be realized as
a protein was first hyphthesized by Franois Jacob and
Jacques Monod.
In 1972, Walter Fiers became the first person actually prove
the existence of the terminating enzyme.
Roger D. Kornberg won the 2006 Nobel Prize in Chemistry for
his studies of the molecular basis of eukaryotic transcription.
Severino Ochoa developed a process for synthesizing RNA in
vitro with polynucleotide phosphorylase, which was useful
for cracking the genetic code.

What is Genetic Engineering?

What are the uses of Genetic Engineering?
What are some genetically engineered food that have
been approved for commercial use?
Is Genetic Engineering beneficial to humans?
How is Genetic Engineering done?
Why is DNA important in Genetic Engineering?
What are the Basics in Genetic Engineering?
History of Genetic Engineering.
What are the Advantages and Disadvantages of
Genetic Engineering?
How long do clones live?

WHAT IS GENETIC ENGINEERING

Genetic engineering (GE) is the modification of an
organisms genetic composition by artificial means, often
involving the transfer of specific traits, or genes, from one
organism into a plant or animal of an entirely different
species. When gene transfer occurs, the resulting organism
is called transgenic or a GMO (genetically modified
organism).
Genetic engineering is different from traditional cross
breeding, where genes can only be exchanged between
closely related species. With genetic engineering, genes
from completely different species can be inserted into one
another. For example, scientists in Taiwan have successfully
inserted jellyfish genes into pigs in order to make them glow
in the dark.

IMPROVES THE LIFESTYLES OF HUMANS

-

Better yield and quality of crops.

Production of super-crops.
Microorganisms as pollution cleaners.
Bioremediation of the polluted environment.

BASICS OF GENETIC ENGINEERING

Different terms used for Genetic Engineering:

WHAT ARE THE USES OF GENETIC ENGINEERING?

1.
2.
3.
4.
5.
6.

To make growth hormone to treat dwarfs.

To prepare vaccines.
To make plants resistant to disease.
To make pigs, cows or fish grow faster.
Higher production of milk by cows.
To make pigs with less fat leaner meat.

WHAT ARE SOME GENETICALLY ENGINEERED FOODS

THAT HAVE BEEN APPROVED FOR COMMERCIAL USE?
-

-Alfalfa
-Cherry Tomato*
-Chicory (Cichorium intybus)
-Corn
-Cotton
-Flax*
-Papaya
-Potato*
-Rapeseed (Canola)
-Rice*
-Soybean
-Squash
-Sugar beet
-Tomato*

IS GENETICALLY ENGINERING BENEFICIAL TO HUMAN?

1.
2.
3.
4.
5.

Gene manipulation
Gene cloning
Recombinant DNA technology
Genetic modification
New Genetics

What is a gene?
A gene is the basic physical and functional unit of heredity.
Genes, which are made up of DNA, act as instructions to
make molecules called proteins. In humans, genes vary in
size from a few hundred DNA bases to more than 2 million
bases. The Human Genome Project has estimated that
humans have between 20,000 and 25,000 genes.
Every person has two copies of each gene, one inherited
from each parent. Most genes are the same in all people, but
a small number of genes (less than 1 percent of the total)
are slightly different between people. Alleles are forms of the
same gene with small differences in their sequence of DNA
bases. These small differences contribute to each persons
unique physical features.
Genes are made up of DNA. Each chromosome contains
many genes.
WHY IS DNA IMPORTANT INGENETRIC ENGINEERING?
DNA is a universal language, meaning the genetic code
means the same thing in all organisms. It would be like if all
cookbooks around the world were written in a single

language that everyone knew. This characteristic is critical

to the success of genetic engineering. When a gene for a
desirable trait is taken from one organism and inserted into
another, it gives the recipient organism the ability to
express that same trait.
HOW IS GENETIC ENGINEERING DONE?
Genetic engineering, also called transformation, works by
physically removing a gene from one organism and inserting
it into another, giving it the ability to express the trait
encoded by that gene. It is like taking a single recipe out of a
cookbook and placing it into another cookbook.

The process: Once a goal is in mind

1) First, find an organism that naturally contains the
desired trait.
2) The DNA is extracted from that organism. This is like
taking out the entire cookbook.
3) The one desired gene (recipe) must be located and
copied from thousands of genes that were extracted.
This is called gene cloning.
4) The gene may be modified slightly to work in a more
desirable way once inside the recipient organism.
5) The new gene(s), called a transgene is delivered into
cells of the recipient organism. This is called
transformation. The most common transformation
technique uses a bacteria that naturally genetically
engineer plants with its own DNA. The transgene is
inserted into the bacteria, which then delivers it into
cells of the organism being engineered.
Another technique, called the gene gun method, shoots
microscopic gold particles coated with copies of the
transgene into cells of the recipient organism. With either
technique, genetic engineers have no control over where
or if the transgene inserts into the genome. As a result, it

takes hundreds of attempts to achieve just a few

transgenic organisms.
6) Once a transgenic organism has been created,
traditional breeding is used to improve the
characteristics of the final product. So genetic
engineering does not eliminate the need for
traditional breeding. It is simply a way to add new
traits to the pool.
HOW LONG DO CLONES LIVE?
Even when cloned animals are born apparently healthy there
are doubts about how quickly they will age.
Results from a number of studies on the lifespan of clones
are beginning to build up. Much of the work so far has been
done on mice because they have relatively short life spans
which makes studying them easier you get results faster
than using cows which may live for twenty years!
In a controlled study almost all of a batch of cloned mice
produced by the National Institute of Infectious Diseases in
Tokyo died earlier than their naturally bred cousins. The
mice all appeared the same when they were young, so
perhaps effects of cloning arent apparent in early life.
Twelve male clones were compared with control groups of 7
naturally conceived males and 6 "test-tube" conceived mice.
Just over 2 years later, 10 of the cloned mice had died
compared to 3 of the non-cloned mice. They had died from a
number of causes including pneumonia, liver disease/cancer
and poor immune systems.
In an experiment with cloned mice at the University of
Hawaii, 1 in 3 clones born looking normal became massively
overweight within a few weeks. There is a gathering amount
of evidence that many clones contain some aspects of their
body systems which dont work well in the long term.

ADVANTAGES
ENGINEERING

AND

DISADVANTAGS

OF

GENETIC

ADVANTAGES
1.

2.

3.

The Capacity of Making Disease a part of History

Humans are considered to be prone in forming one
disease and another. As per the disease, it may
always be the result of bad genes inherited from
parents. Another reason may be attributed on
genetic mutations due to environmental mutagens
that result for diseases such as Alzheimers, cystic
fibrosis and heart diseases. These mutations may
make the body even more susceptible to infections.
A Potential of Increasing the Life Span of People
According to the studies of scientists, genetic
engineering can increase the life span between onehundred to one-hundred fifty years. The technique is
being applied on healthy individuals; changing his
genome which result to slowing down the process of
aging.
Better and Highly-graded Pharmaceutical Products
Genetic engineering acts as an aid for genetics.
Thus, this results to better and Highly-Graded
products that can help humans fight their illnesses
and diseases.

DISADVANTAGES
1.

2.

Genetic engineering is meant to make food crops

more resistant to disease, but the mere act of
modification of the naturally selected food crops
may actually disturb the delicate balance of
biodiversity which exists in nature.
The production of GMOs has negative impacts on
the natural ecosystem which are not apparent
now but will be apparent in the future. For
example, genetic changes in a particular plant or
animal might render it harmful to another

3.

4.

organism higher up in the food chain and

ultimately this effect may build up to destroy the
entire food chain in which that plant plays a role.
GMOs have been known to retain some of the
genetically modified DNA in the final product
made for human consumption. Such remnants of
genetic material are harmful to human health and
can cause production of previously unknown
allergens.
Genetically modified plants and animals have the
potential to replace traditional farming or say
poultry and meat-producing practices. This will
result in destruction of economies based on
these products.

HISTORY OF GENETIC ENGINEERING

1865 Gregor Mendel's publicised his discoveries on the
breeding of peas, which became the foundation of modern
genetics.
1869 Friedrich Miescher discovers nuclein -component of which is DNA -- in the cell nucleus.

major

1999 September, first publicly reported patient death in a

gene therapy trial caused by the gene therapy itself.
1953 James Watson and Francis Crick proposed the double
helix structure of DNA.
1973 Stanley Cohen and Herbert Boyer, invented the
technique of DNA cloning, which allowed genes to be
transplanted between different biological species.
1974 Stanley Cohen, Annie Chang and Herbert Boyer create
the first genetically modified DNA organism.
1980 First transgenic (genetically modified) mouse.
1982 Giant mouse produced by transferring growth hormone
genes from a rat.

1980's to early 1990's China first to put GM crops on sale,

namely a virus-resistant tobacco and a tomato.
1984 Development of genetic fingerprinting, a technique
that has greatly helped the police force in finding and
identifying criminals.

1994 Marking the start of widespread use of genetically

modified crop plants in the USA, the FlavrSavr transgenic
tomato is sold in shops.
1995 A transgenic tobacco variety developed producing
haemoglobin, a human blood protein.

1985 First transgenic domestic animal, a pig.

1996 The birth of the first cloned animal, Dolly the sheep.

1987 A series of transgenic mice produced carrying human

genes.

1996 GM tomato paste approved in the UK, first GM

herbicide tolerant soya beans (Roundup Ready Soybeans)
and insect protected maize approved in the EU.

1988 First transgenic plant producing a pharmaceutical.

1989 Publication (Science 254: 1281-1288) of data about the
'Beltsville pig'; a transgenic pig (named after the agricultural
research station in Maryland USA), which suffered a range of
pathological conditions because it had a gene for human
growth hormone.
1990 GM used to make chymosin, an enzyme used in
making hard cheese.
1991 First gene therapy trials on humans.
1993 U
S Food
and
Drug
Administration
(FDA)
approved Bovine somatotropin (bST) a metabolic protein
hormone used to increase milk production in dairy cows for
commercial use. Scientists determined which gene in cattle
controls or codes for the production of bST. They removed
this
gene
from
cattle
and
inserted
it
into
a
bacterium Escherichia coli. This bacterium produces large
amounts of bST in controlled laboratory conditions. The bST
produced by the bacteria is purified and then injected into
cattle.
1994 Plant IVF
announced.

(in

vitro

fertilisation)

--

maize

(corn).

1997 the cloning of a transgenic lamb (Polly) cloned from

cells engineered with a marker gene and a human gene19
was announced. In this way, the genetic modification of a
lamb was combined with the techniques of cloning, thereby
generating animals that produce a new protein.
2003 Human genome sequenced.
2005 Principles for the European GMO-free regions were
formally laid down in February in Florence during the
Network's 3rd Conference with the subscription of a joint
document called Charter of Florence".
2006 A pig was engineered to produce omega-3 fatty acids
through the insertion of a roundworm gene
2008 The European Commission authorised the GM maize
GA21 for feed and food use and for import and processing.
GA21 is not approved for cultivation in the EU.
2010 Amflora was approved for industrial applications in the
European Union by the European Commission. Amflora is a
genetically modified potato the result of two decades of
research efforts. The Amflora potato is selected for its
special starch properties used in paper making and
adhesives
Term "genetics" was coined by William Bateson in 1905.

1962 Cloning of differentiated (adult) toad cells.

1970 Plants regenerated from protoplasts (plant cells with
cell wall removed).
1979 Twin lambs born through artificial embryo splitting.
1980 First transgenic (genetically modified) mouse.
1982 Giant mouse produced by transferring growth hormone
genes from a rat.
1986 Cloning of embryo cells from sheep.
1952 First successful nuclear transfer.
1997 - Nuclear
laboratory cells

transfer

from

genetically

engineered

1997 - First primate created by embryonic cell nuclear

transfer
1996 - Nuclear transfer from laboratory cells
1987 - Nuclear transfer from embryonic cell

1997 - Two Rhesus Monkeys were cloned by nuclear transfer

from the 8-cell stage in the laboratory of Don Wolf at Oregan
Regional Primate Research Center.
The ewe Dolly (July 5, 1996 February 14, 2003) was the
first animal to be cloned from an adult somatic cell, using
the process of nuclear transfer.
Dolly the sheep, as the first mammal to be cloned from an
adult cell, is by far the world's most famous clone. However,
cloning has existed in nature since the dawn of life.
From asexual bacteria to virgin births in aphids, clones are
all around us and are fundamentally no different to other
organisms. A clone has the same DNA sequence as its
parent
and
so
they
are
genetically
identical.

1984 - First mammal created by nuclear transfer

1975 - First mammalian embryo created by nuclear transfer

1952 - First successful nuclear transfer

1928 - The cell nucleus controls embryonic development
1902 - Artificial embryo twinning in a vertebrate
1885 - First-ever demonstration of artificial embryo twinning
1990 - The Human Genome Project began. This international
collaborative effort attempted to sequence the entire
genetic makeup of humans, consisting of more than 3 billion
nucleotides.

Genetically engineered Pterophyllum Scalara fish glow

in a tank under a blacklight while being displayed at
the 2013 Bio Expo in Taipei, July 18, 2013.
Genetically engineered angelfish (Pterophyllum) glow
in a tank under a blacklight, at a fish farm in Pingtung,
southern Taiwan, September 16, 2010. The fish are
the world's first fluorescent angelfish.
Two transgenic pigs are irradiated under ultraviolet
radiation showing their green fluorescence protein
(GFP) feature at a hogpen in Harbin, northeast China's
Heilongjiang province December 26, 2006. China's
first three transgenic pigs were bred successfully.
Two featherless chickens peck around in some grass
May 22, 2002 at the Hebrew University in Rehovot.
Israeli scientists at the Agriculture department of the
university have genetically engineered bare-skinned
chickens as part of a research project to develop
succulent, low fat poultry that is environmentally
friendly. The naked chicken, as the bird has been
dubbed, would also save poultry farmers large

amounts of money on ventilation to prevent their

chickens from overheating.
Researchers said on April 26, 1999 that they had
genetically engineered goats to produce a human
protein.
Snuppy (R), the first male dog cloned from adult cells
by somatic nuclear cell transfer, and a male Afghan
hound from which an adult skin cell was taken to
clone Snuppy, are seen in this handout photo released
in Seoul, August 3, 2005.
The world's first cloned camel, Injaz (front), is seen at
the Camel Reproduction Centre in Dubai, April 15,
2009. The female camel calf was born on April 8,
created from cells harvested from the ovary of an
adult she-camel which were grown in culture before
being frozen in liquid nitrogen.
Pieraz-Cryozootech-Stallion, a 48 day old cloned foal,
runs in a field outside the northern Italian city of
Cremona April 14, 2005. Pieraz-Cryozootech-Stallion
was born February 25, 2005 from the genes of
castrated endurance champion Pieraz, an Arab
stallion.
A cloned fluorescent puppy, a three-month-old beagle,
is seen with a researcher at Seoul National
University's College of Veterinary Medicine in Seoul
May 13, 2009. The puppy is one of "2nd generation
Ruppies", offspring of "Ruppy", the world's first
transgenic dog which carries fluorescent genes.

DNA TRANSLATION
What is DNA translation?
In molecular biology and genetics, translation is the process
in which cellular ribosomes create proteins. In translation,
messenger RNA (mRNA)produced by transcription from
DNAis decoded by a ribosome to produce a specific amino
acid chain, or polypeptide.

What Is The Function Of DNA Translation?

DNA translation is the process that converts an mRNA
sequence into a string of amino acids that form a protein.
Processes In DNA Translation

Initiation
Elongation
Termination

First Step: Initiation

We'll start with initiation. During initiation, the mRNA, the
tRNA, and the first amino acid all come together within the
ribosome. The mRNA strand remains continuous, but the
true initiation point is the start codon, AUG.
Initiation
Remember that the start codon is the set of three
nucleotides that begins the coded sequence of a gene.
Remember also that the start codon specifies the amino acid
methionine. So, methionine is the name of the amino acid
that is brought into the ribosome first. And how did
methionine get itself to the ribosome? By attaching to the
tRNA that contains the right anticodon. The anticodon for
AUG is UAC. We know that because of the rules of
complementary base pairing.
The tRNA with the anticodon UAC will automatically match to
the codon AUG, bringing the methionine along for the ride.
So, there you have it - mRNA is attached to tRNA, and tRNA
is attached to methionine. That's initiation.
What Is Methionine?
Is an amino acid that is coded by the initiation codon AUG
Second Step: Elongation

A new tRNA+amino acid enters the ribosome, at the next

codon downstream of the AUG codon. If its anticodon
matches the mRNA codon it basepairs and the ribosome can
link the two aminoacids together.
Elongation
(If a tRNA with the wrong anticodon and therefore the wrong
amino acid enters the ribosome, it can not basepair with the
mRNA and is rejected.) The ribosome then moves one triplet
forward and a new tRNA+amino acid can enter the ribosome
and the procedure is repeated.
Third Step: Termination
There are three termination codons that are employed at the
end of a protein-coding sequence in mRNA: UAA, UAG, and
UGA. No tRNAs recognize these codons. Thus, in the place of
these tRNAs, one of several proteins, called release factors,
binds and facilitates release of the mRNA from the ribosome
and subsequent dissociation of the ribosome.

Steps in Cloning
( plasmid, cosmid, virus and YCA)
What is Cloning?
Clones are organisms that are exact genetic copies. Every
single bit of their DNA is identical.
What is DNA Cloning ?

Several steps involved in cloning a gene.

also known as molecular cloning, gene cloning and
recombinant DNA technology - refers to the process of
creating multiple copies of an isolated DNA fragment
or fragments by in vitro or in vivo methods.

How is DNA cloned in cells?

The steps in gene cloning include:

Step 1. The chosen piece of DNA is cut from the source

organism using restriction enzymes.
Step 2. The piece of DNA is pasted into a vector and the
ends of the DNA are joined with the vector DNA by ligation.
Step 3. Transformation of a host cell with the recombinant
DNA(vector DNA with DNA insert)
Step 4: Selection of host cells harboring the recombinant
DNA
Step 5: Screening of cells for those harboring the
recombinant DNA or producing the appropriate protein
product
Cloning Vectors
Cloning Vector
A DNA molecule that carries foreign DNA into a host cell,
replicates inside a bacterial (or yeast) cell and produces
many copies of itself and the foreign DNA.
Bacterial Vector
The greates variety of cloning vectors has been developed
for Ecoli because of the major role they have played in
recombinant DNA experiments since the 1970`s
A cloning Vector Must have:
1. Have an origin of replication so that the DNA can be
replicated within a host cell
2. Be small enough to be isolated without undergoing
degradation during purification
3. have several unique restriction sites for cloning a DNA
fragment so that the vector will be cut only once and several
restriction sites for insertion will be available.

4. Have selectable markers for determining whether the

cloning vehicle has been transferred into cells and to
indicate whether the foreign DNA has been inserted into the
vector.
Three features of all cloning vectors
1.sequences that permit the propagation of itself in bacteria
(or in yeast for YACs)
2.a cloning site to insert foreign DNA; the most versatile
vectors contain a site that can be cut by many restriction
enzymes
3. a method of selecting for bacteria (or yeast for YACs)
containing a vector with foreign DNA; uually accomplished
by selectable markers for drug resistance

2. the tetracycline resistance gene from pSC101

3. The replication region from pMB1
pUC plasmids

Small plasmids containing selection functions that

allow a more direct screening approach, such as the
pUC plasmids developed in 1982
pUC vectors contains the ampicilin resistance
These small vectors (less than 4kb) contain a
polycloning site made up of multiple restriction site,
where foreign DNA can be inserted.
MCS that interrupt -galactosidae gene.
MCS facilitates directional cloning into 2 different
restriciton sites.

Types of Cloning Vectors

pUC18 and pUC19

Plasmid

The pUC18 plasmid and pUC19 plasmid enable successful

cloning of larger DNA fragments than the M13 mp18 RF
Phage Vector. Because these cloning vectors contain a
multiple cloning site at thelacZ' region, recombinant plamids
can be verified via blue/white colony screening using agar
plates containing IPTG and X-Gal. Expression of target DNA
is enabled by the presence of a lac promoter in the cloning
vectors.

Plasmid - an extrachromosomal circular DNA molecule that

autonomously replicates inside the bacterial cell; cloning
limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb)
pBR322 DNA is a commonly used plasmid cloning vector
in E. coli (1). The molecule is a double-stranded circle 4,361*
base pairs in length (2). pBR322 contains the genes for
resistance to ampicillin and tetracycline, and can be
amplified with chloramphenicol. The molecular weight is
2.83 x 106daltons.
pBR322

pBR322 DNA is a commonly used plasmid cloning

vector in E. coli
pBR322 is derived from three naturally occuring
plasmid:

1. the ampicillin resistance gene from plasmid R1.

BACTERIOPHAGE
Viral DNA can be engineered for use as a cloning vector;
lambda bacteriophage. Today many variations of lambda
exist .lambda phage vectors are derived from the 50-kb wildtype ,double-strand genome that has single strand
complementary ends of 12 nucleotides that can base pair.
DNA replication then occurs from the circular molecules
producing linear lambda DNA made up of several 50-kb
phage DNA end on end. In the lytic pathway(cycle)the host
cell lyses after phage reproduction, releasing progeny virus.

Phage
Phage - derivatives of bacteriophage lambda; linear DNA
molecules, whose region can be replaced with foreign DNA
without disrupting its life cycle; cloning limit: 8-20 kb
Cosmids
An extrachromosomal circular DNA molecule that combines
features of plasmids and phage; cloning limit - 35-50 kb
Cosmids are plasmid vectors that contain cos sites.

Problems associated with lambda and cosmid cloning.

Since repeats occur in eukaryotic DNA
rearrangements can occur via recombination of the
repeats present on the DNA inserted into lambda or
cosmid.
Cosmids are difficult to maintain in a bacterial cell
because they are somewhat unstable.

Yeast artificial chromosomes (YACs) are genetically

engineered chromosomes derived from the DNA of the
yeast, Saccharomyces cerevisiae, which is then ligated into
a bacterial plasmid. By inserting large fragments of DNA,
from 1001000 kb, the inserted sequences can be cloned
and physically mapped using a process called chromosome
walking. This is the process that was initially used for the
Human Genome Project, however due to stability issues,
YACs were abandoned for the use of Bacterial artificial
chromosomes (BAC).

A gene is a locus (or region) of DNA that encodes a

functional RNA or protein product, and is the molecular unit
of heredity. The transmission of genes to an organism's
offspring is the basis of the inheritance of phenotypic traits.
Gene Expression
Gene expression is the process by which information from a
gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding
genes such as transfer RNA (tRNA) or small nuclear RNA
(snRNA) genes, the product is a functional RNA. The process
of gene expression is used by all known life - eukaryotes
(including multicellular organisms), prokaryotes (bacteria
and archaea), and utilized by viruses - to generate the
macromolecular machinery for life.
Regulation of gene expression
Regulation of gene expression includes a wide range of
mechanisms that are used by cells to increase or decrease
the production of specific gene products (protein or RNA),
and is informally termed gene regulation. Gene regulation is
essential for viruses, prokaryotes and eukaryotes as it
increases the versatility and adaptability of an organism by
allowing the cell to express protein when needed.

Yeast Artificial Chromosomes (YAC) - an artificial

chromosome that contains telomeres, origin of replication, a
yeast centromere, and a selectable marker for identification
in yeast cells; cloning limit: 100-1000 kb

Regulation of gene expression

Gene

Key Terms:
Operons- an operon is a cluster of coordinately
regulated genes. It includes structural genes,
regulatory genes and regulatory sites.
Lac Operon
Trp Operon
Promoter a nucleotide sequence that enables a gene
to be transcribed. The promoter is recognized by RNA
polymerase, which then initiates transcription.
Operator a segment of DNA that a repressor binds
to. It is classically defined in the lac operon as a

segment between the promoter and the genes of the

operon.
Structural genes the genes that are co-regulated by
the operon.

2 Regulation can be negative or positive

In negative regulation a repressor protein binds to an

operator to prevent a gene from being expressed. (Trp
Operon)
In positive regulation a transcription factor is required
to bind at the promoter in order to enable RNA
polymerase to initiate transcription. (Lac Operon)

What is the importance of Gene Regulation?

Gene regulation is the process of turning genes on and off.
During early development, cells begin to take on specific
functions. Gene regulation ensures that the appropriate
genes are expressed at the proper times. Gene regulation
can also help an organism respond to its environment. Gene
regulation is accomplished by a variety of mechanisms
including chemically modifying genes and using regulatory
proteins to turn genes on or off.
How genes turned on and off?

It all comes down to a process called gene regulation.

This is how our genes are turned off and on, for minor
things like hair color and vital functions like protection
from cancer.
Within our bodies, we house trillions of cells, all busily
going about doing their jobs while we enjoy our days.
Each of those cells has a nucleus that contains our
DNA -- genetic material passed on to us from our
parents. DNA is composed of different sequences of
our genes. These sequences hold directions for
making the proteins that will carry out a cell's
particular function.

This is how one cell might end up being important to your

kidneys, while another cell makes bone.
When a gene is turned off, it no longer provides the
directions for making proteins. This means that the proteins
needed to fulfill a particular job -- say, tolerate lactase -aren't produced. However, when it comes to genes, it isn't a
layer of dirt and metal obstructing the way. It could be one
(or more) of a variety of factors: stages of your
development, the environment, internal influences like
hormones and genetic mutations. Keeping in mind this full
range of factors also helps show that gene regulation isn't
always a bad thing. Just as having to figure out our own
directions every once in a while can be fulfilling to the
explorer in all of us, turning certain genes off and on can be
a completely natural process. Regulation can help our cells
behave properly and aid us in adapting to our environment.
DNA Cloning
What does To Clone mean?

Clone; a collection of molecules or cells.

To clone a gene is to make any copies of it.
Cloned gene can be a normal copy of a gene and an
altered version of a gene.
Recombinant DNA technology makes manipulating
genes possible.

Genetic Engineering
Genetic engineering is the direct manipulation of genes for
practical purposes (DNA fingerprinting, genetically modified
organisms and food, transplantation of genes, cloning)
STEPS IN GENETIC ENGINEERING
Step 1: DNA Extraction
The process of genetic engineering requires the
successful completion of a series of five steps.

DNA extraction is the first step in the genetic

engineering process. In order to work with DNA, scientists
must extract it from the desired organism. A sample of an
organism containing the gene of interest is taken through a
series of steps to remove the DNA.
Step 2 : Gene Cloning
The second step of the genetic engineering process is
gene cloning. During DNA extraction, all of the DNA from the
organism is extracted at once. Scientists use gene cloning to
separate the single gene of interest from the rest of the
genes extracted and make thousands of copies of it.
Step 3 : Gene Design
Once a gene has been cloned, genetic engineers begin
the third step, designing the gene to work once inside a
different organism. This is done in a test tube by cutting the
gene apart with enzymes and replacing gene regions that
have been seperated.
Step 4 : Transformation
Since plants have millions of cells, it would be impossible
to insert a copy of the transgene into every cell. Therefore,
tissue culture is used to propagate masses of
undifferentiated plant cells called callus. These are the cells
to which the new transgene will be added.
The new gene is inserted into some of the cells using
various techniques. Some of the more common methods
include the gene gun, agrobacterium, microfibers, and
electroporation. The main goal of each of these methods is
to transport the new gene(s) and deliver them into the
nucleus of a cell without killing it. Transformed plant cells are
then regenerated into transgenic plants.
The transgenic plants are grown to maturity in greenhouses
and the seed they produce, which has inherited the
transgene, is collected. The genetic engineer's job is now

complete. He/she will hand the transgenic seeds over to a

plant breeder who is responsible for the final step.
Step 5 : Backcross Breeding
The fifth and final part of producing a genetically
engineered crop is backcross breeding. Transgenic plants are
crossed with elite breeding lines using traditional plant
breeding methods to combine the desired traits of elite
parents and the transgene into a single line. The offspring
are repeatedly crossed back to the elite line to obtain a high
yielding transgenic line. The result will be a plant with a yield
potential close to current hybrids that expresses the trait
encoded by the new transgene.
DIFFERENCE BETWEEN CLONING AND GENETIC
ENGINEERING
Cloning
Cloning is a process by which identical copies of an organism
are made. The copy, or clone, possesses exactly the same
genetic material as the original organism. Cloning can occur
naturally through asexual reproduction, wherein a single
organism creates a genetically identical copy of itself.
Bacteria, many plants and even some higher life forms can
reproduce asexually. Scientists have also cloned a variety of
living things, from individual cells to plants and animals. In
addition to Dolly the sheep, cats, dogs, rabbits, deer, mules
and other mammals have been successfully cloned.
Genetic Engineering
Genetic engineering differs from cloning in key ways.
Whereas cloning produces genetically exact copies of
organisms, genetic engineering refers to processes in which
scientists manipulate genes to create purposefully different
versions of organismsand, in some cases, entirely new
living things. Geneticists have even introduced genes from
one species to another. In most instances, the genetically

engineered organism would not occur naturally through

sexual reproduction.
BENEFITS
Cloning and genetic engineering offer benefits, but not all
scientists agree on the ethics and practicality of their
applications. Cloned animals may have uses in medicine; for
example, new drugs could be tested on cloned mice who all
share the same genetic makeup, encouraging a consistent
trial results. In agriculture, chickens that produce eggs at a
faster rate than others could be cloned, thereby increasing
overall egg production. Likewise, researchers can genetically
engineer characteristics in plants or animals to give them
desirable qualities. In the late 1990s, Swiss researchers
created Golden Rice, a strain of rice modified to produce
Vitamin A, a tool for reducing blindness in children across
the developing world.

STEPS IN DNA CLONING

STEPS IN GENE CLONING
1. Isolation of DNA
2. Ligating the DNA into a vector
3. Transformation of a host cell with the recombinant
4. Selection of host cells harboring the recombinant DNA
5. Screening of cells for those harboring the recombinant
DNA or producing appropriate protein.

1. Isolation of DNA
Plasmid is cut open with a restriction enzyme that leaves an
overhang: a sticky end.
2. Ligating the DNA
Bacterial plasmids (small circular DNA additional to a
bacterias regular DNA) are cut with the same restriction
enzyme
3. Transformation
The recombinant plasmids are then mixed with bacteria
which have been treated to make them competent, or
capable of taking in the plasmids
4. Selection of Host Cells
The plasmids have naturally occurring genes for antibiotic
resistance.
Bacteria containing plasmids with these genes will grow on a
medium containing the antibiotic- the others die, so only
transformed bacteria survive

Characteristics of Cloning Vectors

It should be able to replicate autonomously.

Origin of replication.
Selectable markers.
Restriction sites.

PLASMID pbr322
Contains:
Selectable Markers:
Ampicillin resistance gene.
Tetracycline resistance gene.
Col E I replication origin.
Eco RI site.
Puc

Plasmids

Contains

Ampicillin resistance gene.

Multiple cloning site.
ColEI ( origin ).

5. Screening of Cells

Yeast Artificial Chromosome

The transformed bacterial cells form colonies on the medium

A linear chromosome, has centromere, telomeres, ARS

(autonomously replicating sequence), selectable marker for
yeast (uracil or tryptophan biosynthesis genes usually).

Each cell in a given colony has the same plasmid (& the
same DNA)
Cells in different colonies have different plasmids (&
different DNA fragments)
CLONING VECTORS
A cloning vector is a DNA molecule in which foreign DNA can
be inserted or integrated and which is further capable of
replicating within host cell to produce multiple clones of
recombinant DNA.

Also has E. coli ori and selectable marker: you can grow the
vector itself in E. coli
Then purify it, ligate in foreign DNA, transform into yeast.
COSMIDS
Are plasmid vectors that contain cos sites. The cos site is the
only requirement for DNA to be packaged into a phage
particle. Cosmids were developed in light of this observation
How do you clone into cosmid vectors?

1. Clone the DNA into the vector as you would with any
plasmid.
2. Introduce the DNA into the bacterial cell via a phage
particle.
3. Propagate as plasmid.
4. Since phage particles can accept between 38 and 53
kb of DNA and since most cosmids are about 5 kb,
between 33 and 48 kb of DNA can cloned in these
vectors.
Problems associated with lambda and cosmid cloning.

Since repeats occur in eukaryotic DNA

rearrangements can occur via recombination of the
repeats present on the DNA inserted into lambda or
cosmid.

Cosmids are difficult to maintain in a bacterial cell

because they are somewhat unstable.

Features of YACs
Large DNA (>100 kb) is ligated between two arms. Each arm
ends with a yeasttelomere so that the product can be
stabilized in the yeast cell. Interestingly, larger YACs are
more stable than shorter ones, which favors cloning of large
stretches of DNA.
One arm contains an autonomous replication sequence
(ARS), a centromere (CEN) and aselectable marker (trp1).
The other arm contains a second selectable marker (ura3).