The control of gene expression

Gene Mutations

Recap
We have seen how:

DNA makes RNA makes Protein

We know that the specific DNA
coding, makes a specific mRNA
coding which translates to a specific
amino acid sequence in the protein.

What is a Mutation?

A mutation is any change in the amount or

structure of the DNA of an organism.
KEY POINT:
If this occurs in somatic (body) cells, the
change cannot be inherited. Only mutations
in the DNA within gametes can be passed on
to the next generation.

Causes of Mutations

Mutations occur naturally and at random.

However, mutagens are environmental factors
that increase rate of mutation.
High-energy
radiation

High-energy
particles from
radioactive
substances

Mutagenic
chemicals

Carcinogenic
substances such
as tar and
asbestos

Gene Mutations

Point Mutations

These are point mutations involving a change of only

one nucleotide at a particular locus. There are 3
types:
1. Substitution
One nucleotide is replaced by another with a different
base.
2. Addition
An extra nucleotide is added so an extra base is
added to the sequence.
3. Deletion
One nucleotide is removed.

Substitutions

Substitutions only affect one codon in a

sequence of genetic material. They therefore
only affect the outcome of a single amino acid.

G C T A G T
A AT AC G T
Arginine

Serine
Tyrosine

Tyrosine

Alanine

The above animation is a worst case scenario when it

comes to substitution mutations.
Sickle Cell
Anaemia

Why might a substitution mutation not have an

effect on protein shape at all?

 Remember the genetic code is

degenerate.
That means that most amino acids
have more than one codon.
Therefore, a substitution mutation
has no effect (usually) on the
resulting polypeptide that forms.

Addition

When an extra base is added into the

sequence. This results in a frame shift and
the whole sequence of triplets is altered.
This frame shift occurs to the RIGHT.

G C T A G A AT AC G T

T
G C T A G A AT AC G T
G C T T A G AA T A C G

Some extra points about addition

mutations
If three bases (or any multiple of
three bases) are added as mutations,
there will be no frame shift.
The resulting polypeptide will be
different from the one that is
normally produced, however, not as
different as one resulting from a
frame shift.

Deletions

Deletions also result in frame shift, but this

time to the LEFT.
As all the triplets will be different, this will
result in different amino acids, and therefore
a different polypeptide.

G C T A G A AT AC G T
G C T A G A AT AC G T
G C A G A A T A CG T T

Other types of mutations

Duplication when one or more bases are
repeated. This results in a frame shift to the
right.
Inversion- when a group of bases dissociate
from the DNA sequence, but then rejoin at the
same place BUT in a back to front order.
Translocation when a group of bases from a
DNA sequence on one chromosome detach and
reattach onto another different chromosome.

Homework
Attempt the past paper questions on
gene mutations.
Finish the rest off for homework.
Bring in for tomorrows lesson please
to go through and mark.

Stem Cells and

Totipotency

Learning Objectives

Learn about totipotency of cells.

Learn about when totipotent cells are present
and what their role is.
Learn how these cells can lose their
totipotency.
Learn about multipotent cells.
Learn how totipotent and multipotent cells can
be used to treat human disorders.

Stem Cells

TOTIPOTENCY

Growth and Development

Growth of an organism is a simple

concept, but development is much more
complicated especially in organisms as
complex as mammals.
Development involves the specialisation of
cells and arranging them into
functional
This Blue
Whale has grown
to an enormous size.
units.
Its development though, is
a series of changes
involving cell
differentiation.

It contains....
2 x 1017 cells....
200,000,000,000,000,000....
200 quintillion!
Each one is specialised.

Growth and Development

Development involves producing

specialised cells and arranging them into
tissues and organs.
Because cells in different areas of the body
have to carry out specific functions, they
have to become adapted.
The process of becoming adapted is known
as cellular differentiation.
An unspecialised cell becoming a
muscle cell

To understand this better, you need to think back to the

first cell that ever had anything to do with your
existence...

This cell is the

precursor for all the
cells in your body at
this moment in time.
This cell will divide
countless times,
giving rise to more and
more daughter cells,
until there are the 75
or so trillion cells that
make up... you.
This one cell, has all
of the information
required to produce
all of the millions of

O
O
D
A
H
!
W SH
When a sperm and an
egg cell fuse, the
resulting cells is called a
zygote.

zygote

morula (approx 16
cells)

For the first few

weeks, the
developing
embryo contains
unspecialised
cells.

Embryonic stem
cells are highly
important
because they
can
differentiate
into any type of
blastocyst
cell.
We call
these...
embryo
nic stem
cells

Youre too specialised

In an adult, all cells are specialised.

They have already been through the process
of differentiation and cannot transform into
another type of cell.
Only embryonic stem
cells have the capacity
to become any type
of cell.
We call this ability....

totipotency

Totipotency

tot
alp
i otential
Totipotent cells have the ability to develop
into any cell found in the human body.
This is obviously useful during embryo
development, as we start off as a single cell,
but are eventually a collection of millions of
different cell types.
But what decides which stem cells in an
embryo go on to become a certain cell
type?

Here is a
totipotent cell

Imagine it was taken from a

very simple mammal, with
only a single homologous
chromosome pair
heart cell gene

It was taken
from an
embryo

That DNA in those csomes

contains genes
(instructions) to make any
cell type from any organ.

intestinal cell gene

brain cell gene

The lineage that the stem cell takes, depends on

which genes within it, are expressed.
If genes related to heart structure are expressed, the
stem cell will become a heart cell.
Once a stem cell differentiates, it can never go back
to being totipotent.

Unused library

Most of the DNA in a cell is never even transcribed!

E.g.: If a cell differentiates into a liver cell, it will
never need to translate any mRNA that is not
involved in liver activities.
It still has DNA in it that is related to every other
cell in the body, but the only special gene it may
ever need to transcribe, could be the one for
insulin.
In summary: Totipotent stem cells are
unspecialised. They occur briefly in embryos.
They can differentiate into any type of cell. Once
they differentiate, they lose their totipotency.

Taking a specific route...

A developing embryo contains entirely

totipotent cells for the first few weeks of its
existence.
Eventually,
the cells in the embryo
begin to specialise, each diverting
into its own lineage.
i.e. One totipotent stem cell will
take up the responsibility for the
digestive system.
But we know the digestive system is complicated... It
contains many different tissues and organs.
The cell that took up the responsibility for this system,
has now got to divide and specialise into individual
sections of the digestive system (e.g. Pancreas,
small intestine etc.)

Stem Cells

MULTIPOTENCY

Animal cells are one-hit-wonders...

Youve learnt that embryos contain totipotent
stem cells with unlimited potential...
Once a cell becomes specialised, although it
still contains all the genes of the organism,
the specialisation is irreversible.
That is to say,
for example....
A cell of the
liver, can now
not become
(or divide
into) a cell of
the brain.

Except for a select few...

Although the majority of cells in your body lost
their totipotency while you were an embryo....
There are a select few types of cell in
your body that can still differentiate to a
certain extent.
They are called
MULTIPOTENT CELLS
(or adult stem cells).

These dont have the

totipotency of
embryonic stem cells,
but are still able to
differentiate.
They can only

Bone marrow is the site where blood cells are

manufactured. But because there are different types of
blood cell, they are produced from multipotent stem
cells.

Stem Cells

USING STEM CELLS TO

TREAT DISORDERS

Stem Cells in Medicine

Stems are used in medicine to cure some diseases.
Leukaemia is cancer of the blood. The blood cells
grow out of control, and abnormally. With
abnormal white blood cells, a person with
leukaemia cant fight off disease.
Because bone marrow contains stem
cell that can differentiate into
blood cells.....
.....bone marrow transplants can
help a patient with leukaemia
develop healthy blood cells.

Stem cells from adult bone

How about stem cells from an embryo?

Stem cells from a developing embryo can develop
into any cell type....
......so a patient with paralysis or brain damage
could benefit
if stem cells could be instructed to grow into
new nerve cells.A person who has suffered
extreme burns could benefit
from skin grafts...
.... the skin cells would be
developed from embryo stem
cells.

The embryo would be

sacrificed in order to improve
a life. Is this acceptable?

Ethics of Stem Cell Research

Each embryo is a potential human life. So
some people argue that it is unacceptable to
experiment with embryos.
But some people think that it is better to end a
persons suffering, rather than debate an
embryos right to existence.
When do you think an embryo becomes a
human?

7 days
newborn baby

9 weeks

Unwanted embryos
An unwanted pregnancy, may be terminated by
abortion.
This means the embryo/foetus is extracted
from the womb prematurely.
A lot of countries allow
abortion, but are
unwilling to conduct
stem cell research.
This embryo was
terminated at 7 weeks.

You can see that stem cell research is a

very important, but controversial topic in
science.

Umbilical cords
Using embryos for stem cell research is bound to
raise ethical issues.
However, scientists have discovered that the
umbilical cords of newborn babies contain a
reservoir of stem cells, which is ethically
much more acceptable.
Cord blood is collected,
which contains a number
of different stem cell
types, some of which can
be manipulated to be
made totipotent.
As of yet, their uses are
still limited.

Regulation of Transcription
& Translation

Learning Objectives
Learn what a transcription factor is.
Learn how oestrogen affects gene
transcription.
Learn what siRNA is and how it
affects gene expression.

Recap
That the cells in our bodies are highly specialised.
They have specific functions to perform in
different areas of the body, and have structures that
reflect these functions.
Essentially, what are all structures in cells made
of?

PROTEIN
In order to produce these molecules, what
process did we establish had to occur?

GENE EXPRESSION
Gene expression is just a fancy way of saying.....
some DNA is used to produce protein.

Here is a
totipotent cell

Imagine it was taken from a

very simple mammal, with
only a single homologous
chromosome pair
heart cell gene

It was taken
from an
embryo

That DNA in those csomes

contains genes
(instructions) to make any
cell type from any organ.

intestinal cell gene

brain cell gene

The lineage that the stem cell takes, depends on

which genes within it, are expressed.
If genes related to heart structure are expressed, the
stem cell will become a heart cell.
Once a stem cell differentiates, it can never go back
to being totipotent.

GENE EXPRESSION

Gene Expression
You know that the basics of gene expression is that:
1. Transcription has to occur.
2. Pre-mRNA has to be spliced.
3. Translation has to occur.
But what decides
when this happens
and at which section
of DNA?!

Its all well and good knowing the process of getting

from gene to protein product, but how is this
process regulated?
Does it just happen?

Transcription Factors
Genes dont just start to transcribe themselves
spontaneously.
If that was the case, cells in your pancreas would produce
adrenaline, and cells in testicles would begin to release
oestrogen!

Your body contains regulatory proteins called

TRANSCRIPTION
DNA Binding FACTORS.
Transcription
factors are a
protein
complex, with
different
subunits.

Site

Transcriptio
n Factor

Receptor

Hormone
Binding Site

How do the Transcription Factors Work?

The gene that codes for the required protein, is
stimulated by a specific transcription factor.
There are millions of transcription factors and
each one has a DNA binding site that is
specific to a certain gene.
When it binds to the correct region of DNA,
transcription begins.
This would then produce mRNA, which would
then be translated into a protein.

What about when the gene

doesnt need to be expressed?
How could you stop
transcription factors from
stimulating DNA?

There are 2 possibilities if you think about it....

1.

....maybe not

2.

Inhibitor
Molecule

When a gene is not

being expressed, the
DNA binding site on
its complimentary
transcription
factor is BLOCKED.
This inhibitor stops
the transcription
factor from binding
to DNA, thus
blocking
transcription from

HORMONES

There are 2 Mechanisms of Hormone

Action

The first mechanism involves protein hormones

(such as insulin) and molecules called second
messengers.
Transcription and translation though, are
regulated via the other hormone mechanism,
which involves lipid-soluble hormones (such as
oestrogen).
Protein
Lipid-Soluble
Hormones

Hormones

Act via Second

Messengers

Act Directly

e.g. Insulin

e.g. Oestrogen

Hormones like oestrogen can switch

on a gene and start transcription.
They do this by binding to their
receptor on the transcription factor.
This changes the transcription
factors shape, and thus releases
the inhibitor molecule.
The transcription factor can then bind
to DNA, starting up the process of
transcription.

Using siRNA to Prevent

Gene Expression

siRNA
Gene expression can be prevented by breaking
down mRNA before it is translated into a protein.
To do this, small molecules of double-stranded RNA
called siRNA are essential (small interfering RNA).
Large double-stranded molecules are cut into siRNA
by enzymes.
The siRNA splits into single-stranded molecules, of
which one, associated with a different enzyme.
The siRNA guides this enzyme to an mRNA molecule.
Once there, the enzyme cuts the mRNA into small
sections.
This renders the mRNA useless, as transcription
cannot occur.

siRNA inhibits translation of mRNA and

turns genes OFF
Enzyme 1
breaks up
dsRNA making
siRNA
molecules
Enzyme 2
combines
with one of
the two
molecules of
siRNA
Complimentary
base pairing
between siRNA
and target
mRNA

Enzyme 2 cuts
mRNA into small
sections stopping
it from being
translated

Uses of siRNA
1. It could be used to identify the role of genes in a
biological pathway. By using siRNA to block
certain genes, you could observe what effects
occur. This could then tell you what the role of
the blocked gene is.

2. Some diseases are genetic and are caused by

the expression of certain genes. If these
genes could be blocked by siRNA, it may be
possible to prevent the diseases caused by
them.

Epigenetic Control of
Gene Expression

https://www.youtube.com/wa
tch?v=_aAhcNjmvhc

What are epigenetic markers and

what do they do?
They are chemical groups that
can attach or remove
themselves to DNA or histones.

They do not
alter DNA bases
sequences

They determine
whether or not genes
are expressed.

Epigenetic
markers

They alter how easy it is for

enzymes needed for
transcription to interact with
DNA and then to transcribe it.

Epigenetic changes can occur in

normal cellular processes, and can
occur in response to environmental
changes (eg pollution or
availability of food)

 Histones are proteins that

DNA wraps around to
form chromatin.
Chromatin makes up
chromosomes.
Chromatin can be highly
or less condensed.
How condensed it is
affects the accessibility of
the DNA and whether or
not it can be transcribed.

When acetyl groups

attach to histones,
chromatin becomes
less condensed.
This means the
transcription
machinery can easily
access the DNA, and
therefore genes are
expressed.
When acetyl groups
are removed from
histones, chromatin
becomes more
condensed.
This means the
transcription
machinery cannot
easily access the
DNA, and therefore
genes are not

Cancer
Know how the rate of cell division is controlled by tumour
suppressor genes and proto-oncogenes
Explain the effects of a mutation on tumour suppressor
genes and proto-oncogenes

Be able to interpret information relating to the use of

oncogenes and tumour suppressor genes in the prevention
and cure of cancer
Be able to evaluate the effect on diagnosis and treatment
of disorders caused by hereditary mutations and those
caused by acquired mutations

What is Cancer?
Mutations that occur in cells after
fertilisation are called acquired
mutations.
If these mutations occur in the genes that
control the rate of mitosis, it can cause
uncontrolled cell division.
If a cell divides uncontrollably, a mass of
abnormal cells will form- this is a tumour.
Tumours that invade and damage
surrounding tissues are called
cancers.

There are two types of gene that

control cell division. Mutations in these
genes can cause cancer.

Homework task:
Due 18th November 2016
Compare the differences between
malignant and benign tumours.
Aim for at least 8 different
points.

They dont
produce all the
proteins in order
to function
properly.

They divide
more
frequently
than normal
cells.

Different
antigens on
their cell
surface

Tumour cells
look and
function
differently
to normal
cells

They do not respond

to growth
regulating

Irregular
shape

Larger and
darker
nucleus than
usual

Methylation

Why do you think cervical and stomach cancers are

more common in LEDCs?

Metastasis

Locating and Sequencing

Genes

Learning Objectives
Recap how DNA probes and DNA
hybridisation is used to locate
specific genes.
Learn how the exact order of
nucleotides on a strand of DNA can
be determined.
Learn how restriction mapping can
be used to determine nucleotide

DNA Probes

DNA probes are simple, short and singlestranded sections of DNA.

They will bind to complementary sections of
other DNA strands.
Due to being labelled in some way, they make
this other DNA easily identifiable.
Labelling with
radioactivity

Labelling with
fluorescence

Remember that probes can be used as

an easy method of screening
(detecting) for mutated genes.
But also remember that the probe
needs to be complementary to the
mutated gene.
So this means, that to produce a probe,
you first need to sequence your gene.
How do we sequence genes?

Meet Frederick Sanger...

Biochemist
Cambridge University
English
Two Nobel Prizes
Still Alive

Sangers work in the 1970s,

which earned him his second
Nobel Prize, involved the
sequencing of DNA.
His method used modified nucleotides that do
now allow another nucleotide to join after them in
a sequence.

Sanger Sequencing Method

Introducing Sanger Sequencing

The method is based on the premature ending

of DNA synthesis.
If modified nucleotides are used during DNA
synthesis, the process can be halted.
What normally happens during DNA synthesis...

T A T G G A T C T G A C C T T A G
A T A C C T A G A C T G G A A T C
What happens if you modify a nucleotide...

You call these modified

nucleotides,
T A T G G A T C
TERMINATORS
A T A C C T A G A C T G G A A T C

What you need...

In Sanger Sequencing, four different terminators

are used (A, C, T and G).
Due to this, four different reactions are run.

In each reaction, you have the following:

A
T

G
C

A
C

The DNA being sequenced.

A mixture of normal nucleotides (A, T,
C, D)
One type of terminator nucleotide.
A primer.
DNA Polymerase.

A
T

A
C

A
T

A
C

Remember that each tube probably contains millions of copies of

the DNA template, countless nucleotides, and a good supply of
the specific terminator nucleotide.
Due to this, you get a variety of partially completed DNA strands,
because they have been terminated at different points.

So what happens in each tube?

Lets take the example of the tube with an

adenine terminator
Now lets imagine this is the sequence of
the unknown DNA strand:

CCGTCTAGCACTCAAGC
TCT

What are the possible

terminated sequences going
to be when the reaction is
over?

G
A

A
C

GGC
GGC
GGC
GGC
C
GG
GA
C

A
A
A
A
A

Because there are both normal

and terminator nucleotides in the
A mixture, there is a chance that
either is placed as the next base

G
GATCGTGA
GATCGTGAGTT
GATCGTGAGTT

Remember that this is happening in

four test-tubes, each with a different
type of terminator nucleotide.
DNA fragments in each of the four
tubes are going to be of varying
lengths.
Now the lengths of DNA need to
be separated, so that we can see
why we went through all of this
trouble...

GEL ELECTROPHORESIS

Gel Electrophoresis

When youve got a mess of DNA, especially DNA

strands of varying lengths, you can separate them out
using this technique.
The whole process relies on the fact that the
phosphates in the backbone of
DNA,
are negatively
DNA
fragments
are placed
charged.
in wells at the top of an

agar gel.
An electric current is
applied over it.
Agar is actually a mesh,
which resists the movement
of the DNA fragments
through it.
The DNA moves towards the
positive electrode, but at
different rates.
Small sections get there
quicker.

Back to Sanger Sequencing

The fragments produced during the reactions can be

separated using gel electrophoresis.
The smallest fragments will move furthest along the
gel in a fixed period of time.
Due to being radioactively labelled, we can see
where the DNA fragments end up, by placing
photographic film over the gel, after the run.
Termina
tor C

Termina
tor A

Termina
tor T

Termina
tor G

Automated Sequencing

Nowadays, DNA sequencing is automated, using

computers.
Nucleotides are fluorescently labelled with dyes.
Everything occurs in only a single tube.
And the separation can occur in one lane during
gel electrophoresis.

Equipment used
during the Human
Genome Project.

Flash Video
http://smcg.ccg.unam.mx/enp
-unam/03-EstructuraDelGenom
a/animaciones/secuencia.swf

Recombinant DNA
technology
Recombinant DNA technology involves the transfer of
fragments of DNA from one organism, or species, to
another, resulting in translation within the recipient
(transgenic organism) due to the universal nature of the
genetic code.

Fragments of DNA can be produced by several methods,

including:
conversion of mRNA to cDNA, using reverse transcriptase
using restriction enzymes to cut a fragment containing
the desired gene from DNA
creating the gene in a gene machine

Diagram showing
rDNA production

Use of Recombinant DNA

Technology. (Genetic engineering)
Looking at how genetic modification has
benefitted humans and what roles GM plants
and animals have had in the beneficial use
of recombinant DNA technology.

What is genetic
engineering?
Having looked at how to form DNA
fragments and clone them to useable
quantities it is now time to look at their
uses and how they can benefit humans.
Organisms that have had their DNA altered
by genetic engineering are called
transformed organisms.
These organisms have recombinant DNA
DNA formed by joining together DNA from
different sources.

Genetically modified
microorganisms.
Transformed microorganisms can be made using the same
technology as in vivo cloning e.g. foreign DNA can be inserted
into microorganisms to produce lots of useful proteins e.g.
insulin:
1. The DNA fragment containing the insulin gene is isolated
(using reverse transcriptase/restriction endonuclease/PCR)
2. The DNA fragment is inserted into a plasmid vector using
ligases - ligation.
3. The plasmid containing recombinant DNA is transferred into
a bacterium
4. Transformed bacteria are identified and grown marker
genes/fluorescence.
5. The insulin produced from the cloned gene is extracted and
purified.

 Antibiotics are also produced naturally by bacteria.

Genetic engineering has increased the quantity
and rate at which these bacteria can do so.
Enzymes used in the food industry are
manufactured by genetically modified bacteria to
produce large quantities for less money. For
example rennin is an enzyme used in cheese
making. It used to be made from rennet (a
substance produced in the stomach of cows) but it
can now be produced from transformed organisms.
This means it can be made in large quantities
relatively cheaply without killing any cows and
making the cheese suitable for vegetarians.

Social, moral and ethical concerns of

the use of GM microorganisms.
Without proper labelling some people
think that they wont have a choice
about whether to consume food
made using GM organisms.
Some people are worried that the
process used to purify proteins from
GM organisms could lead to the
introduction of toxins into the food
industry.

GM plants and agriculture.

Agricultural crops can be transformed so that they
have higher yields or are more nutritious. This
means that these plants can be used to reduce the
risk of famine and malnutrition. Crops can also be
transformed to have pest resistance so that fewer
pesticides are needed. This reduces costs and any
environmental problems associated with pesticide
use.
E.g. see text book pg. 257 and also:
Golden rice is a variety of transformed rice. It
contains one gene from a daffodil plant and one
gene from a soil bacterium which together enable
the rice to produce beta carotene. The beta
carotene is used by our bodies to make vitamin A
and so can be used in areas where there is a

Social, moral and ethical concerns

over GM plants
Farmers might plant only one type of
transformed crop making the whole crop
vulnerable to disease because the plants are
genetically identical.
Some people are concerned about the
possibility of superweeds weeds that are
resistant to herbicides. These could occur if
transformed crops interbreed with wild plants.
Concern by some people over what they are
actually eating as the long term effects of
consuming GM food are unknown.

GM animals.
Animals can be genetically modified
so as to increase their growth rate
and as such reduce the cost of
rearing them before sending to
market.
Animals can also be made to be
disease resistant making
domesticated animals more
economical to rear and reducing
overall food costs.

Social, ethical and moral issues of

GM animals
Many people fear that Genetically
modifying animals is cruel and only
encourages intensive farming
methods which can lead to very
stressful environments for animals.

GM and medicine.
Many drugs and vaccines are produced by
transformed organisms using recombinant DNA
technology. They can be made quickly and cheaply
and in large quantities.
E.g. insulin used to treat type 1 diabetes used to
come from cow/pig/horse pancreases and It didnt
work quite as well. It is now produced using
microorganisms to get a pure human product (see
earlier).
Rare and expensive proteins can also be made
.make notes on the production of anti-thrombin in
goats from page 257 fig 2 page 258 is useful.

Ethical, moral and social concerns

over GM use in medicine.
Companies who own Genetic
engineering technologies may limit
the use of technologies that could be
saving lives.
Some people worry this technology
could be used unethically to produce
designer babies. This is currently
illegal.

Balancing the arguments.

One of the requirements of this topic
is to be able to balance the
humanitarian benefits of GM
technology with opposing views from
environmentalists and anti
globalisation activists.

Potential humanitarian benefits of

genetic engineering.
1. Agricultural crops could be produced to help
reduce the risk of famine and malnutrition
e.g. drought resistant crops.
2. Transformed crops could be used to produce
useful pharmaceutical products such as
vaccines. This could make drugs available to
more people e.g. in areas where refrigeration
needed for storing vaccines isnt available.
3. Medicines could be produced more cheaply
so more people can afford them.

Environmentalists and antiglobalisation activists have concerns

1. Environmentalists oppose recombinant DNA technology
because they think it could potentially harm the
environment e.g. transformed crops could encourage
farmers to carry out monoculture which decreases
biodiversity. There are also fears that if transformed
crops breed with wild plants theyll be uncontrolled
spread of recombinant DNA with unknown consequences.
2. Anti-globalisation activists these are people who oppose
globalisation. A few large biotechnology companies
control some forms of genetic engineering. As the use of
this technology increases, these companies get bigger
and more powerful. This may force smaller companies
out of business by making it harder for them to compete.

Question
1. A large agricultural company has isolated
a gene from bacteria that may increase
the drought resistance of wheat plants.
2. A) briefly explain how this gene could be
used to make a transformed wheat plant
(3)
B) Suggest how the transformed wheat
plants might be beneficial to humans (2)
C) Suggest why anti-globalisation activists
may be against the use of this gene (1)

Gene therapy & Cystic

fibrosis

Recap
Do you know what each of these
are?
Plasmids
Phospholipid bilayer
Recessive alleles
Carriers

Do you know:
How we cut & join DNA?
Why we use viruses in genetic
engineering?

Cystic Fibrosis - what is it?

Cystic fibrosis is the most common
inherited disease. 1 in 20 people are
carriers of the recessive allele that
causes it. 1 in 2000 people have the
disease.
The carriers are perfectly healthy,
but if two carriers have children the
chances of any of their children
having the disease is
1 in 4.

Cystic Fibrosis - what is it?

The disease is caused by a recessive allele on
chromosome 7. The gene codes for a
transmembrane regulator protein (an ion pump)
called CFTR which is present in epithelial cells in
the body. CFTR pumps chloride ions across the
membrane and water molecules follow these
ions out of the cell; this keeps the epithelial cells
moist and smooth.
But in cystic fibrosis patients, the gene (cfcf) has
a different base sequence and either codes for
no protein or for a faulty one (which can be
lacking just 1 amino acid). As a result the
epithelia remains dry and a thick mucus builds
up.

Adiagramofthehumanlargeairways.Oneofthemostcompellingreasonsto
attemptgenetherapyforCFwasthattheairwaysshouldpresentarelatively
simple,easytoreachtarget.Intruththeairwayspresentmanymorebarriersto
successfulgenetherapythanmostotherorgans.

Protein channels

Healthy CFTR
channel
proteins

CFTR channel
proteins in cf
sufferer

Cystic Fibrosis what can we do about it?

How could we prevent, treat or cure CF? Brainstorm
under these headings
Prevention

Treatment

Cure

Cystic Fibrosis what can we do about it?

Prevention

Screening parents (spit test)

Genetic guarantees
Screening foetuses (then aborting if positive)
Genocide or sterilising carriers

Treatment
Enzyme pills (for digestion)
Physiotherapy & massage (to remove mucus)
Gene therapy - putting copies of the healthy gene where
they are needed

Cure
Gene therapy - changing all the affected cells are
permanently altered
Altering the zygote (fertilised egg) with genes

Gene therapy
Gene therapy means
using genes as a
treatment or a cure. In
most cases it means
putting copies (clones)
of a healthy gene where
they are needed to
replace faulty genes.
What would this mean
for cystic fibrosis?

Problems with gene therapy

1.
2.
3.
4.

Isolating the relevant gene

Getting the gene to the right cell
Getting the gene into the right cell
Getting the gene to work in the cell

You have already covered part 1, we

will look at how to do the last 3 bits.

http://www.cfgenetherapy.org.uk/clinical/multidose.html

http://www.cfgenetherapy.org.uk/research/viral.html

Two methods of treatment

The following are being tried, but as
yet neither is approved for widescale use:
Liposome sprays
Viral sprays

These treatments last only until the

epithelial cells are routinely replaced,
so they would have to be regularly
repeated. Both treatments can be
given either by a simple hand-held
puffer (like those asthmatics use) or
through a nebuliser.

Liposomes sprays
The normal CFTR gene is inserted
into a bacterial plasmid. Once
cloned by bacteria the copies of the
plasmids with the CFTR gene are
made into liposomes (tiny lipid
bilayer droplets)
An aerosol spray of these liposomes
is used to introduce the genes to the
lungs. The liposomes fuse with the
cell membrane lipids and the gene is
delivered into the epithelial cell.
The gene leads to the functioning
pump CFTR being made in the cell.

Problems
It is difficult to get a fine enough
spray to get the liposomes through
the bronchioles to the alveoli.
Only a small number of the genes
that get absorbed are actually
expressed.

Viral sprays
Genetic code can be programmed into
cells in the lungs through use of
aerosols. Copies of the non faulty gene
are inserted into a virus (an
adenovirus) especially modified so
that it can infect the lungs but not
reproduce. The virus gets into the lung
cells and injects its modified genes into
the cell.
This makes the cells produce the CFTR
protein thus reducing the symptoms of
cystic fibrosis.

Adenovirus

Problems
There are concerns that the virus might
cause an infection itself! This could
happen if the genes that allow the virus to
replicate have not been totally inactivated.
It is possible that the patient could
develop antibodies making them immune
to the virus - this would ruin the
treatment!

Problems with gene therapy

Alongside ethical issues there are other
problems:
The effect is short-lived so continued
therapy is needed
It can induce an immune response
meaning the treatment is rejected
Using viral vectors presents problems
might cause disease or a toxic response
from the recipient
The genes are not always expressed
It is not effective in treating conditions
caused by a fault in more than one gene
(e.g. arthritis or Alzheimers)

In vivo gene cloning the

use of vectors

The importance of sticky ends.

Last lesson, we discussed sticky ends that are left
after the action of restriction endonucleases.
These are highly important in genetic engineering,
for the fact that they leave exposed bases this
is due to the staggered nature of the cut.

Due to the complementary base-pairing rules of

DNA, sticky ends on one gene, will pair up with
sticky ends of another bit of DNA that has also
been cut by the same restriction endonuclease.

This gene (from a human) can

be cut with a restriction
enzymesuch as EcoRI
Sticky End

KEY:
Gene from
Human
Gene from E.coli

This is a section of DNA from

E.coli.

Sticky End

If this section
of DNA from
E.coli is also
cut with
EcoRI, a
complimenta
ry sticky end
is produced.

If these two cut pieces of DNA

are mixed, recombinant DNA

Once the bases have paired, they form

their usual weak hydrogen bonds
between each other.
The only thing left to do, is form the
link between the sugar-phosphate
backbones, and this is done by the
enzyme, DNA Ligase.

Inserting genes into Plasmids

The real-life application of what we have just
learnt, occurs when geneticists insert an animal
or plant
gene
into plasmids.
This
is Step
2 (insertion) in the
Plasmids
are small
of DNA
which are
process
ofloops
making
a protein
found in addition to the large circular
using gene technology
chromosome that bacterial cells possess.
By inserting our chosen gene into a plasmid, the
plasmid acts as a carrier, or vector, which we
Restriction
can
then introduce back
into a bacterial cell.
Endonuclease
Restriction
Endonuclease
DNA coding for a
desired protein
Remember, that
DNA Ligase would
once again be used
to bond the sugar-

A plasmid
As the DNA fragment was cut
out using the same restriction
endonuclease as used to cut
the plasmid open, they have
complimentary sticky ends.

Introducing our recombinant plasmids into

host cells

Introducing recombinant plasmids into bacterial

cells is called transformation.
This is done by mixing the plasmids with the cells
in a medium containing calcium ions.
The calcium ions make the bacterial cells
permeable, allowing the plasmids to pass
through, into the cell.
However, only a few
bacterial cells (approx 1%)
will actually take up the
plasmids.
Calcium ion medium

For this reason, we need to

identify which ones have
been successful. This is
done with gene markers.

This is Step 3 (transformation) of

producing a protein by DNA technology

Using Gene Markers to identify successful

host cells...
There are a number of different ways of using gene
markers to identify whether a gene has been taken up
by bacterial cells.
They all involve using a second, separate gene on the
plasmid. This second gene is easily identifiable for one
reason or another....
It may be resistant to an antibiotic
It may make a fluorescent protein that is easily
seen
It may produce an enzyme whose action can be
identified
Each of the 3 above mechanisms are actual methods of
employing gene markers to identify bacterial cells that have
taken up plasmids... They will be discussed on the next
slides...

Enzyme Markers

Fluorescent
Markers

Gene
marke
rs
Antibiotic-resistant
Markers

1. Antibiotic-Resistance Markers
Many bacteria contain antibiotic resistance genes in
their plasmids. Some in fact, can have two genes
for resistance to two different antibiotics, in the
Any bacterial cell posessing this
same plasmid. Gene for
plasmid, would be resistant to

Gene for
resistance
to
ampicillin

resistance
to
tetracycli
ne

both of the antibiotics,

ampicillin and tetracycline.
But what if we cut right in
the middle of the
tetracycline-resistance gene
(with a restriction
endonuclease), and insert a
with this
gene of our Bacteria
own interest?
plasmid would only
be resistant to
ampicillin, not
tetracycline.
How is this of any
advantage to us?

First, the recombinant plasmids are

introduced into bacterial host cells
(transformation)

The bacteria is grown on

agar treated with
ampicillin

Colonies are
allowed to
grow, but will
only do so if
they are
resistant to
ampicillin i.e.
Bacteria that
A replica plate is now made. This is when you literally
took press
up the
the agar of one Petri-dish, onto the agar of a new Petri-dish,
plasmid.
transferring bacterial cells from each colony onto the new agar.

This agar
however,
has been
treated
with
tetracyc
line

?
Colonies are allowed to
develop

There is a
missing
colony, which
has lost
resistance to
tetracycline.
This must be
a colony
containing

2. Fluorescent Markers
This is a more recent method of finding out whether
bacteria have taken up the desired plasmids.
Throughout nature, there are organisms
such as jellyfish, that produce
fluorescent proteins.
These are proteins, which obviously
have their own genes, which of course
can be isolated and then introduced into
bacterial cells via vectors.
The range of natural fluorescent proteins
can be seen on this Petri-dish. These are
bacteria
are insert
expressing
colonies
All you of
have
to dothat
is first
your gene of interest (such as
the
fluorescence
insulin)
into
a gene forgenes!
a fluorescent protein.

Then insert this insulin/fluorescence hybrid gene into a plasmid

vector.
Then transfer the plasmids into bacterial cells!
Any cells that successfully took up plasmids, will be glowing on your
Petri-dish!

3. Enzyme Markers
This method involves inserting your gene of interest
(e.g. Insulin), into a gene that codes for an enzyme
such as lactase.
There is a particular substrate that is usually
colourless, but turns blue when lactase acts upon it.
If you insert you chosen gene into the gene that
makes lactase, you will inactivate the lactase
gene.
If you now grow bacterial cells on an agar medium
containing the colourless substrate, any colonies that
have taken up the plasmid, will not be able to change
its colour to blue.
Any colourless spots will indicate to you, which cells
have been transformed.
The more boring of the 3 methods, but still important.

In vitro cloning
Polymerase Chain Reaction

Objectives
You should be able to describe the
biological principles underlying each
of the following:
The use of the polymerase chain
reaction (PCR) to make large
amounts of DNA from very small
samples
The use of genetic fingerprinting

Polymerase Chain
Reaction(PCR)

PCR is a method by which DNA can

be replicated in the lab.
It can be used to create millions of
copies of DNA in just a few hours.
It is essential in forensic science as
very small samples of DNA are
difficult to analyse.
This process amplifies DNA, so that it
can be analysed.

What do you need?

1. RNA primers provide the starting
sequence for DNA replication. They
also stop the two DNA strands from
joining together.
2. DNA nucleotides containing the
bases adenine, guanine, cytosine
and thymine.
3. Enzyme DNA polymerase.

The Stages of PCR

Strand Separation
DNA heated at
95C for 5mins

Mix with
Primers (RNA
C
strands)

Binding of
Primers
Mixture cooled
to 40C
Mix with
Free Nucleotides
DNA Polymerase

REPEAT
CYCLING
With every cycle the amount
of DNA doubles

DNA Synthesis
Mixture heated
to 70C
(optimum temp.
for DNA
polymerase)

The Double Stranded DNA

Molecule

A G G

A G

G A

C C

Heat to 950C to separate the DNA

strands

DNA Strand

A G G

C C

RNA Primers

C
T

A G

G A

DNA Strand
Cool to 400C to allow primers to bind
(anneal) to DNA

DNA Polymerase

A
T

Original DNA
strand

A G G

A G

Primer

C C

Nucleotides join
on DNA
Free

A
T

C C

Nucleotides join
on

nucleotides

A G

A
T

Primer

G A

Original DNA
strand

T
A

Free DNA
nucleotide
s

Mix with DNA polymerase and free nucleotides and

0

Advantages
It is a very rapid process
Does not require living cells
Is useful when we want to introduce
a gene into another organism

Disadvantages
These are also the advantages of In vivo
cloning:
There is risk of contamination by
unwanted DNA
It cannot cut out specific genes
Does not produce transformed bacteria

Summary Questions
In the polymerase chain reaction, what are
the primers?
What is the role of these primers?
Why are two different primers required?
When DNA strands are separated in the PCR,
what type of bond is broken?
It is important in the PCR that the fragments
of DNA used are not contaminated with any
other biological material. Suggest a reason
why.

DNA HYBRIDISATION
One technique which enables scientists to carry out a DNA comparison is calledDNA hybridisation. This
involves the joining together of DNA sections from 2 species to be compared. These are the steps involved:

1. DNA samples are collected, cut into smaller sections, then heated to 90 degrees.

3. The separated strands from the two species are now put together and allowed to cool.

4. Some strands will join back with their original pair; others will join with strands from the other organism
to formhybrid DNA.

The temperature at which these strandsre-anneal(bond together) is the clue to the genetic relationship
between the two organisms. DNA strands which joined back with their original counterpart from the same
organism re-anneal at 87 degrees, as they share a lot of base sequences. Thehybrid DNA, on the other
hand, is formed at alower temperature. This is because fewer sequences are shared, and so fewer
hydrogen bonds are formed which hold the strands together.

The lower the temperature at which hybrid DNA forms between two organisms, the less genetically
related they are. This technique has led to a new classification system being used for plants.

2. The heat denatures the DNA molecules by breaking thehydrogenbonds within; the strands of DNA
separate.