AQA Biology A-level

Topic 8: The control of gene expression

Notes

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Mutations
Mutations are changes in the sequence of nucleotides in DNA molecules. Types of mutations
include:

• Insertion/deletion mutations - where one or more nucleotide pairs are inserted or

deleted from the sequence. This type of mutation alters the sequence of nucleotides
after the insertion/deletion point known as a frameshift.

• Duplication - one or more bases are repeated and therefore produces a frameshift.

• Inversion - a group of bases become separated from the DNA sequence and then rejoin at
the same position but in the reverse order. This therefore affects the amino acid that is
produced.

• Translocation - a group of bases become separated from the DNA sequence on one
chromosome and are inserted into the DNA sequence on another chromosome. This can
often lead to significant effects on the phenotype.

Cause of mutations

Gene mutations can arise spontaneously during DNA replication, and can be caused by
mutagenic agents that affect DNA, causes of gene mutations are:

1. Chemical mutagens - these include alcohol, benzene and substances in asbestos and is tar in
tobacco.
2. Ionising radiation - alpha and beta, but also UV and X-ray.
3. Spontaneous errors in DNA replication

Mutations can either have neutral effects where the mutation causes no change to the
organism, for example in a case where the mutation occurs in a non-coding region of DNA or is a
silent mutation. A mutation can also be neutral when a change in tertiary structure of the
protein has no effect on the organism.

Some mutations are beneficial, for instance, humans developed trichromatic vision through a
mutation. Harmful mutations include a mutation in the CFTR protein which causes cystic
fibrosis.

Stem cells
Stem cells are undifferentiated cells which can keep dividing to give rise to other cell types.
Types of stem cells include pluripotent cells that are able to give rise to many types of
specialised cells apart from embryonic cells and totipotent cells which can give rise to all
types of specialised cells including embryonic cells.

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Totipotent stem cells that are able to differentiate into any type of cell found in body and into
extra embryonic cells such as those in the placenta. These cells are found in the embryo at an
early stage called the blastomere . These stem cells are sometimes called embryonic stem
cells.

The totipotent cells in the embryo are initially unspecialised however when they become
specialised they differentiate to form tissues which make up the foetus. The cause of this is a
change in gene expression where some genes are selectivity switched on and others switched
off.

There are a variety of different types of stem cells and are named according to their ability to
differentiate. They are summarised below:

1. Totipotent - can form any type of cells in the body plus extra embryonic cells.
2. Pluripotent - these cells can form any cell type in the body, however cannot form extra
embryonic cells. They are also found in the early stages of an embryo. These are often used in
replacing damaged tissues in human disorders.
3. Multipotent - can differentiate into other cells
types but are more limited e.g. the cells in
the bone marrow and umbilical cord.
4. Unipotent - these cells can only differentiate
into one type of cell.

Pluripotent cells also have a number of different

uses in repairing damaged tissue, these are
shown in the table.

Pluripotent stem cells can also be created from

unipotent stem cells and are therefore known as
induced pluripotent stem cells (iPS).

Regulation of transcription and translation

Control by oestrogen

The hormone oestrogen has the ability to alter transcription through altering molecules called
transcription factors. These are molecules that bind to a specific site on DNA to begin the
process of transcription. The action of oestrogen in controlling transcription is described below:

1. The lipid soluble nature of oestrogen means that is can freely diffuse across the cell
membrane where is binds to a receptor molecule on a transcription factor.
2. The binding alters the shape of the DNA binding site on the transcription factor and makes it
able to bind to the DNA.
3. The transcription factor therefore enters the nucleus via the nuclear pore where it binds to
DNA. This stimulates the transcription of the gene that makes up the DNA.

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Small interfering RNA

Small interfering RNA (siRNA) also called silencing RNA is used for short term switching off of
genes. The siRNA binds to a complementary sequence of mRNA. As mRNA is usually single
stranded and the cell therefore detects the double stranded form on mRNA and views it as
abnormal. Therefore the mRNA is broken down by enzymes preventing translation.

Epigenetic changes

• Epigenetics involves heritable changes in gene function, without changes to the base
sequence of DNA. It shows that environmental factors can make changes to the function
of genes which can be inherited.

• DNA methylation is a process by which methyl groups are added to DNA. Methylation
modifies the function of the DNA, typically acting to suppress gene transcription. DNA
methylation alters the expression of genes in cells as they divide and become specialised.
The change is permanent and prevents the cell from converting back into a stem cell or a
different cell type. The methylation is through the addition of a CH₃ chemical group to
cytosine bases, which both prevents binding of transcriptional factors to DNA and
stimulates decreased acetylation of histones.

• DNA acetylation also changes DNA structure. Histones are positively charged proteins
closely associated with DNA, which is negatively charged. Decreased acetylation of
histones increases their positive change, so they bind DNA more tightly. When this
happens, transcriptional factors can no longer access the DNA, so the gene is switched
off.

Gene expression and cancer

Cancer can arise as a result of mutation. Uncontrolled cell division in cancer leads to the
formation of a tumour. There are two types of tumours, benign which do not tend to cause
much harm apart from light mechanical damage caused by pressing against blood vessels or
other cells. Benign tumours grow slowly and do not spread, whereas malignant tumours grow
rapidly and can spread to the neighbouring cells via metastasis (through the blood stream or
lymphatic system) thus causing damage by disrupting the running of important processes.
Malignant tumours are difficult to treat in comparison to benign tumours.

The following play an important role in cancer:

• Proto-oncogenes – stimulate cells to divide by producing proteins that stimulate cell

division, allow the checkpoints of the cell cycle to be passed, and can cause cancer if
mutated.

• Oncogenes - these are formed from mutated proto-oncogenes and result are
permanently switched on resulting in cell division that is uncontrolled. It does this by
permanently activating a cell surface receptor or coding for a growth factor.

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 • Tumour suppressor genes – control cell division, cause the cell cycle to stop when
damage is detected. They also play a role in the programming of apoptosis (cell death).
When these are switched off the cell cycle becomes unregulated.

• Abnormal methylation of tumour suppressor genes and oncogenes - increased

methylation also called hyper-methylation plays an important role in controlling tumour
suppressor genes and oncogenes. The hyper-methylation of a tumour suppressor gene
called BRAC1 can lead to breast cancer.

• Increased oestrogen concentrations can be linked to breast cancer development. These

elevated levels are found in fatty tissues called adipose tissue in the breast of post-
menopausal women. Oestrogen binds to the transcription factor which activates the
genes promoting cell division, leading to tumour formation.

Genome projects
Sequencing projects have read the genomes of a wide range of organisms, including humans.
Determining the genome of simpler organisms allows the sequences of the proteins that derive
from the genetic code of the organism to be determined. This may have many applications,
including the identification of potential antigens for use in vaccine production. In more
complex organisms, the presence of non-coding DNA and of regulatory genes means that
knowledge of the genome cannot easily be translated into the proteome. The proteome is all
the proteins that the genome can code for. However due to selective gene expression not all
of these proteins will be found in every cell in the body.

Gene sequencing allows for genome-wide comparisons between individuals and between
species. Comparing genomes between species is significant as it allows evolutionary
relationships between species to be determined, and it is also beneficial to medical research.
Comparing genomes of individuals enables differences to be identified which can then be used
for development of personalised medicine tailored to a particular genome, as well as in
studies of human diseases.

Apart from allowing genome-wide comparisons to be made, gene sequencing has allowed for the
sequences of amino acids in polypeptides to be predicted and has allowed for the
development of synthetic biology.

The Human Genome Project is an international scientific project which has successfully
determined the sequence of bases of a human genome. Potential applications include:
screening for mutated sequences, carriers and pre-implantation screening as well as
screening for disorders such as Huntington’s disease before the symptoms appear. However,
there are many ethical concerns regarding the Human Genome Project, such as people being
discriminated against as well as regarding the misuse and ownership of the genetic information.

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Recombinant DNA technology
Recombinant DNA technology involves many ways of manipulating DNA, these processes are all
detailed below.

Using reverse transcriptase to make DNA

The enzyme reverse transcriptase is a enzyme that is found in only some viruses and bacteria
and catalyses the formation of a double strand of DNA from a single strand on RNA. This
allows us to make working versions of DNA that act as genes, by extracting mRNA from cells
where that gene is being expressed.

Using restriction endonucleases to cut DNA fragments

Restriction endonucleases are enzymes,

extracted from bacteria, that cut DNA at
specific sequences, usually six base pairs
in length. The most useful restriction
endonucleases are those that make
staggered cuts, as they leave sticky ends
on the DNA. The digram shows the
difference between sticky ends and blunt
ends.

Sticky ends are important because if the

same restriction endonuclease is used to cut two DNA fragments then the ends will be
complementary. This allows then to attach together before stronger covalent bonds form.

In-vivo gene cloning

If a DNA fragment was placed in a cell it would be digested

by enzymes and therefore a vector is used to insert DNA
into cells. They are the plasmids from bacterial cells that
naturally occur.

Isolated DNA fragments can be placed in plasmids in a

following way:

• Plasmid and gene are cut with the same restriction

enzyme to create complementary ends sticky ends.
This means that they inserted DNA and vector are
complementary and can be joined.
• The fragments are incubated with the plasmids. If a
plasmid takes up the insert, base pairing takes place
between the complementary ends which are then
sealed with the use of DNA ligase which forms
phosphodiester linkages.
• A recombinant DNA molecule is created

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In the formation of transgenic microorganisms, electroporation is used to stimulate bacterial
cells to take up plasmids. Electroporation facilitates the process by increasing the permeability
of bacterial membranes thus increasing the chance of success. This is achieved via the use of
calcium salts and rapid temperature change from 0 to 40 degrees.

Gene markers

In order to check whether the DNA has been taken up by the bacteria gene markers are used.
There are different types of gene markers, these are antibiotic restraint genes, fluorescent
markers and enzyme markers. These genes are incorporated into the plasmid so that those
who have the plasmid can be separated from the bacteria that do not.

They are also used to determine whether the desired DNA has enter the plasmid, as the marker
gene will become inactivated.

Gene technologies
DNA profiling is a forensic technique used to identify individuals by characteristics of their
DNA. It can also be used to determine genetic relationships between organisms. One example
is PCR.

Polymerase chain reaction known as PCR is used to amplify DNA by making millions of copies of
a given DNA sample. It occurs as following:

1) A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides
and DNA polymerase which is the enzyme involved in creating new DNA strands.
2) The mixture is then heated to 95 degrees to break the hydrogen bonds and to
separate the two strands.
3) The mixture is then cooled to a temperature between 50-65 degrees depending on
the type of primers used, so that they can bind (anneal) to the strands.
4) Temperature is increased to about 70 degrees as this is the temperature DNA
polymerase works at. The DNA polymerase is called Taq polymerase and is from
bacteria that live in hot springs.

5) DNA polymerase creates a copy of the sample by complementary base pairing using
the free nucleotides.
6) This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient
to create a DNA profile.

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In-vivo and in-vitro cloning
In-vitro - this gene cloning can be done
with PCR. This is fast, automated and
reliable once conditions are established.
This does not require living cells and can
have problems such as contamination and
errors.
In-vivo - gene cloning that can be done
using recombinant plasmids in bacteria.
This is accurate and useful as the gene is
placed in cells where it can be expressed.
The disadvantage though is it is very time
consuming and requires monitoring of
cell growth.

DNA probes
A DNA probe is a short, single stranded
DNA molecule that is designed to
complementary to a sequence to be
detected. DNA probes are made in smaller
quantities and then amplified using PCR.
The DNA labelling of the fragments either
uses radioactive isotopes or a fluorescent
dye which glows under certain
wavelengths of light.
DNA probes can be used in order to detect
heritable conditions of health risks. The
diagrams shows the process.

Genetic fingerprinting
Genetic fingerprinting is a technique that can detect differences in people DNA. It uses
variable number tandem repeats (VNTRs) which are short repeating sequences or bases. The
probability of two individuals having identical VNTRs is extremely low therefore VNTR analysis
can be used in genetic fingerprinting.

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Gel electrophoresis is a process used to
separate the DNA fragments and proteins
according to their size using an electric
current. It occurs as shown in the digram on the
right.

Genetic fingerprinting can be used in the fields

of forensic science, medical diagnosis as well as
animal and plant breeding. It is carried out as
follows as shown in the diagram below.

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