Cell & Molecular Biology

Course Outline
• This course involves a detailed study of 4 Units:
• (1) Cell and Molecular Biology
• (2) Environmental Biology
• (3) Physiology, Health & Exercise
• (4) Investigation
• There is practical work within units (1) - (3), while unit
(d) is based on research and experimentation entirely.

Overview
• UNIT 1: Cell and Molecular Biology
- The structure & function of prokaryotic and eukaryotic cells
- The structure and function of cell components
- Molecular interaction in cell events
- Applications of DNA technology
• UNIT 2: Environmental Biology
- Circulation in ecosystems
- Interaction in ecosystems
- Human impact on the environment
• HALF UNIT 3: Physiology, Health and Exercise
- Exercise and the cardiovascular system
- Exercise and metabolism
• HALF UNIT 4: Investigation
- A scientific investigation into a syllabus-related topic that
is designed, carried out and written up as a 2000-2500 word
report, worth 20% of the final marks.

Methods of Learning
• Powerpoint presentations, directed reading and
laboratory work
• Practical work emphasises experimental skills used to
investigate basic issues associated with each topic
• You are also expected to devote considerable effort on
the planning, evaluation and writing up of your
investigations

Homework
• Homework is set on a regular weekly basis. At least 3
hours per week in self study at home!
- Short essay type answers like those in sections C and D
of the external examination
- Data Handling questions like those in sections B, C and D
of the external examination and unit tests
- Writing up learning outcomes to produce summaries
- Revision for unit assessments, topic tests etc.
- Planning, carrying out and writing up the investigation
- Writing up class practical activities

Internal Assessment
• End of unit tests
Unit awards are obtained by taking a NAB at level C
only. 65% is required to pass the test
• Experimental reports
You will carry out, record and write up, in the form of a
report, one experiment. To achieve a course award in
the final examination, all three unit tests and the
experimental report must be passed
• End of topic tests
A/B tests are taken at the end of every unit and
prelims are held during the year also

External Assessment
2 1/2 hour external examination with a total of 100
marks (worth 80%)
SECTION A 25 Multiple choice
Units 1 and 2 only
25 marks
SECTION B 7 questions
Short/Extended/data qu’s
Units 1 and 2 only
55 marks
SECTION C 4 questions
Short answer
Unit 3 only
20 marks

Career Opportunities
• A good Advanced Higher pass will enhance an application for
courses at College or University level, especially of
Biological subjects … any course really (it shows that you
have an excellent brain!)
• In terms of University admission, Advanced Higher Biology
is rated equivalent to A Level Biology, but the investigative
research in Advanced Higher provides a very valuable
opportunity at this level of study

Advanced Higher Biology
UNIT 1
Cell & Molecular Biology

STRUCTURE, FUNCTION &
GROWTH OF PROKARYOTIC &
EUKARYOTIC CELLS

Prokaryotic and Eukaryotic Cells
• All living creatures are
made up of CELLS, small
membrane bound units
filled with aqueous
solutions of chemicals,
which have the ability to
create copies of
themselves by growing and
dividing.
[The sizes of cells and organelles]

• Living organisms can be classified into 3 major domains:
Bacteria
Archaea
Plant cells
Animal cells
• Prokaryotes and Eukaryotes are 2 distinct cell types
with STRUCTURAL differences
PROKARYOTES
EUKARYOTES

The Prokaryotic Cell
• Simply stated,
prokaryotes are
molecules surrounded by
a membrane and cell wall.
1 um

Prokaryotes
• Lack a membrane bound nucleus enclosing the DNA
• DNA is present as a single circular molecule called a BACTERIAL
CHROMOSOME
• DNA is naked having no associated histone proteins
• No membrane bound organelles
• Apart from the DNA nucleoid, there is little internal structure apart
from dissolved substances and a large number of RIBOSOMES essential
for PROTEIN SYNTHESIS
• The cytosol is an effective site for bacterial cell metabolism. This allows
bacteria to adapt quickly to changing nutritional conditions, but means
the regulation of genetic and metabolic activity has to be tightly
regulated.
• Divide by BINARY FISSION
• Some prokaryotic cells have external whip-like FLAGELLA for locomotion
or hair like PILI for adhesion.
• Prokaryotic cells come in multiple shapes: cocci (round), baccilli (rods),
and spirilla or spirochetes (helical cells).

External Prokaryotic Structures
Cell Wall
• Contains PEPTIDOGLYCAN (only
found in bacteria). Large complex
molecule consisting of
polysaccharide polymers cross-
linked by short chains of amino
acids
Capsules
• Sometimes the cell wall is
further surrounded by a
gelatinous polysaccharide sheath
called an attach CAPSULE,
GLYCOCALYX or SLIME LAYER
Plasma Membrane
• Basic structure of the
phospholipid bilayer is the same
for all bacteria
Flagella
Motile bacteria usually have long,
thin appendages called FLAGELLA.
These protein sub-units are used to
propel bacteria through liquids

Pili or Fimbrae
• A pilus (Latin; plural : pili) is a
hairlike protein structure on
the surface of a bacterial cell,
required for bacterial
conjugation (transfer of
genetic material)
• A fimbrium (Latin; plural:
fimbria) is a short pilus that is
used to attach the cell to a
surface. Mutant bacteria that
lack fimbria cannot adhere to
their usual target surfaces
and, thus, cannot cause
diseases.

Spores & Cysts
These are produced by some bacteria to survive
unfavourable environmental conditions. Dormant forms are
metabolically inactive and only germinate under suitable
conditions
ENDOSPORES: a dormant, tough, non-reproductive
structure produced by a small number of bacteria. The
primary function of most endospores is to ensure the
survival of a bacterium through periods of environmental
stress. They are therefore resistant to ultraviolet and
gamma radiation, desiccation, lysozyme, temperature,
starvation, and chemical disinfectants. Endospores are
commonly found in soil and water, where they may survive
for long periods of time e.g. Clostridium (tetanus, gas
gangrene), Bacillus (anthrax)
CYSTS: also dormant, but unlike endospores are not
resistant to heating at high temperatures

Classifying Prokarotes
• Main method is using the GRAM’S STAIN
• This separates bacteria into GRAM-POSITIVE (purple) and
GRAM-NEGATIVE (red) depending on the percentage of
PEPTIDOGLYCAN in the cell walls
- GRAM-POSITIVE bacteria have a cell wall only 1 layer thick
- GRAM-NEGATIVE bacteria have a cell wall several layers thick

Eukaryotes
• More complex multicellular organisms e.g. plants,
animals, fungi and also many single-celled organisms e.g.
amoeba, yeast
• Possess an NUCLEUS and other organelles all of which
are surrounded by a MEMBRANE, which divided the cell
up into compartments
COMPARTMENTALISATION: very important !
ADVANTAGES:
•Molecules are ‘concentrated’ together, increases rate of reactions
•Keeps reactive molecules away from other parts of the cell that may be affected by them
•Large work surface area … many enzymes are bound in membranes

Eukaryotes
The basic eukaryotic cell contains the following:
- membrane-bound nucleus
- plasma membrane
- glycocalyx (components external to the plasma
membrane)
- cytoplasm (semifluid)
- cytoskeleton – microfilaments, intermediate filaments and
microtubules that suspend organelles, give
shape, and allow motion
- presence of characteristic membrane
enclosed subcellular organelles e.g.
mitochondria, golgi, rER, sER etc

Plant & Animal Cells
• For ANIMAL CELLS only:
– Peroxisomes & Lysosomes often present
– Some have microvilli on their surface
– Centrioles organise spindle fibres during cell division
• For PLANT CELLS only:
– Cell walls made from cellulose
– Communication with neighbouring cells occurs through
plasmodesmata
– Usually a large central vacuole
– Photosynthesis occurs in cells containing chloroplasts
[Stick in & label plant & animal cell diags]

Plasma Membrane
Plasma Membrane
A lipid/protein/carbohydrate
complex, providing a barrier and
containing transport and
signalling systems.

Nucleus
Nucleus
Double membrane surrounding
the chromosomes and the
nucleolus. Pores allow specific
communication with the
cytoplasm. The nucleolus is a
site for synthesis of RNA
making up the ribosome

Mitochondria
Mitochondria
• Surrounded by a double
membrane with a series of
folds called cristae.
• Functions in energy
production through
metabolism.
• Contains its own DNA, and is
believed to have originated as
a captured bacterium.

Rough endoplasmic reticulum (RER)
Rough endoplasmic reticulum (RER)
• A network of interconnected
membranes forming channels within
the cell.
• Covered with ribosomes (causing the
"rough" appearance) which are in the
process of synthesizing proteins for
secretion or localization in
membranes.
Ribosomes
• Protein and RNA complex responsible
for protein synthesis

Golgi Apparatus
Golgi apparatus
• A series of stacked membranes.
Vesicles (small membrane surrounded
bags) carry materials from the RER to
the Golgi apparatus.
• Vesicles move between the stacks while
the proteins are "processed" to a
mature form.
• Vesicles then carry newly formed
membrane and secreted proteins to
their final destinations including
secretion or membrane localisation.

Centrioles
Centrioles
• Centrioles are found only in
animal cells. They function in
cell division.

Lysosymes
Lysosymes
• A membrane bound organelle
that is responsible for
degrading proteins and
membranes in the cell, and
also helps degrade materials
ingested by the cell.

Peroxisomes
Peroxisomes or Microbodies
• Produce and degrade hydrogen
peroxide, a toxic compound that
can be produced during
metabolism

Chloroplasts
Chloroplasts
• Surrounded by a double
membrane, containing stacked
thylakoid membranes.
• Responsible for photosynthesis,
the trapping of light energy for
the synthesis of sugars.
• Contains DNA, and like
mitochondria is believed to have
originated as a captured
bacterium.

Vacuoles
Vacuoles
• Membrane surrounded "bags"
that contain water and
storage materials in plants.

Cell wall
Cell wall
• Plants have a rigid cell wall in
addition to their cell
membranes. They provide
support for the plant.

Similarities between P & E cells
• Prokaryotes & Eukaryotes are CHEMICALLY & METABOLICALLY
similar:
– Both have genetic material
– Both have a cell membrane
– Both have a cytosol
– Both have ribosomes
– Both contain nucleic acids, proteins, carbohydrates & lipids
– Both use similar reactions for storing energy and metabolic
activities e.g. building proteins

Differences between P & E cells
• Main differences are STRUCTURAL:
PROKARYOTES EUKARYOTES
No membrane bound nucleus Membrane bound nucleus
Cell walls made of peptidoglycan
(Thickness of wall depends on whether the
cell is Gram +ve or –ve)
Cell walls, if present, made of cellulose
(chitin in fungi)
No membrane bound organelles Membrane bound organelles
(compartmentalisation)
Have pili & fimbriae (for adhesion) and
flagella (for propulsion)
Have cilia or flagella (for movement)
Mucilaginous capsule No mucilaginous capsule present
(numerous internal structures present
including microtubules, ER, Golgi, secretory
vesicles etc)
Cell size ranges from 0.5um to 100um Cell size ranges from 10 – 150um

Comparison of Prokaryotic and Eukaryotic Cells
PROKARYOTES EUKARYOTES
Organisms
Monera: Eubacteria and
Archebacteria
Protists, Fungi, Plants and Animals
Level of
organization
single celled
single celled (protists mostly) or
multicellular usually with tissues and
organs
Typical cell size small (1 -10 microns) large (10 - 100 microns)
Cell wall almost all have cell walls (murein)
fungi and plants (cellulose and chitin);
none in animals
Organelles usually none
many different ones with specialized
functions
Metabolism anaerobic and aerobic; diverse mostly aerobic
Genetic
material
single circular double stranded
DNA
complex chromosomes usually in pairs;
each with a single double stranded DNA
molecule and associated proteins
contained in a nucleus
Mode of
division
binary fission mostly; budding
mitosis and meiosis using a spindle;
followed by cytokinesis

Cell Growth & The Cell Cycle
• Living things can be
distinguished from
non-living things by
their ability to
REPRODUCE
• This characteristic
is based on cells
being ability to
DIVIDE

What is DNA and
where is it stored?
• The nucleus is a membrane
bound organelle that contains
the genetic information in the
form of chromatin, highly
folded ribbon-like complexes
of deoxyribonucleic acid
(DNA) and a class of proteins
called histones.

Cell Cycle
• Cell division allows organisms to grow, develop, to rweplace dead
cells and to repair tissue
• This is a CONTINUAL PROCESS
• The length of the cell cycle depends on the type of cell and
external factors e.g. temp, O2 supply etc
• Bacterial cells – 20 mins
• Liver cells divide only once a year or only if the need arises e.g.
injury
• Skin cells – all the time
• Nerve & muscle cells don’t divide at all in a mature adult

The Cell Cycle
• Stages in the Cell Cycle:

Cell Growth and The Cell Cycle
• A eukaryotic cell cannot divide into two, the two into
four, etc. unless two processes alternate:
– doubling of its genome (DNA) in S phase (synthesis
phase) of the cell cycle;
– halving of that genome during mitosis (M phase)
• The period between M and S is called G1; that between
S and M is G2.

• For a new cell to be produced …
– The quantity of DNA must double – DNA replication
– Must be copied EXACTLY
Due to the brief flurry of cytological activity during
cell division, the cycle is divided up into 2 parts:
INTERPHASE (G1, S, G2 phases)
MITOTIC PHASE (M phase)

So, the cell cycle consists of:
• G1 = growth and preparation of the
chromosomes for replication
• S = synthesis of DNA (and centrosomes)
• G2 = preparation for
• M = mitosis
• When a cell is in any phase of the cell cycle other than
mitosis, it is often said to be in Interphase.

What is (and is not) mitosis?
• Mitosis is nuclear division plus cytokinesis, and
produces two identical daughter cells during prophase,
metaphase, anaphase, and telophase.
• Interphase is often included in discussions of mitosis,
but interphase is technically not part of mitosis, but
rather encompasses stages G1, S, and G2 of the cell
cycle.

Interphase • The cell is engaged in metabolic
activity and performing its
preparation for mitosis (the next
four phases that lead up to and
include nuclear division).
• Chromosomes are not clearly
discerned in the nucleus, although a
dark spot called the nucleolus may be
visible.
• The cell may contain a pair of
centrioles (or microtubule organising
centres in plants) both of which are
organisational sites for microtubules.

Prophase • Chromatin in the nucleus begins to
condense and becomes visible in the light
microscope as chromosomes.
• The nucleolus disappears. Centrioles begin
moving to opposite ends of the cell and
fibres extend from the centromeres.
• Some fibres cross the cell to form the
mitotic spindle.
• The nuclear membrane dissolves, marking
the beginning of metaphase.
• Proteins attach to the centromeres
creating the kinetochores. Microtubules
attach at the kinetochores and the
chromosomes begin moving.

Metaphase
• Spindle fibres align the
chromosomes along the middle of
the cell nucleus. This line is
referred to as the metaphase
plate.
• This organisation helps to ensure
that in the next phase, when the
chromosomes are separated, each
new nucleus will receive one copy
of each chromosome.

Anaphase
• 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.

Telophase
• 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 fibres disperse,
and cytokinesis or the
splitting of the cell may also
begin during this stage.

Cytokinesis
• In animal cells, cytokinesis
results when a fibre ring
composed of a protein called
actin around the centre 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
synthesised between the two
daughter cells.

Remember …!
• Prophase
• Metaphase
• Anaphase
• Telophase
• Cytokinesis
• Positive
• Mental
• Attitude
• Towards
• Calvin Klein

DNA Replication
• Before a cell can divide, it must duplicate all its DNA. In
eukaryotes, this occurs during S phase of the cell cycle.
• Recap the steps in DNA replication ….
• A portion of the double helix is unwound by a helicase.
• A molecule of a DNA polymerase binds to one strand of the DNA
and begins moving along it in the 3' to 5' direction, using it as a
template for assembling a leading strand of nucleotides and
reforming a double helix.
• Because DNA synthesis can only occur 5' to 3', a molecule of a
second type of DNA polymerase binds to the other template
strand as the double helix opens. This molecule must synthesize
discontinuous segments of polynucleotides (called Okazaki
fragments). Another enzyme, DNA ligase I then stitches these
together into the lagging strand.

DNA Replication is Semiconservative
• When the replication process is
complete, two DNA molecules —
identical to each other and
identical to the original — have
been produced. Each strand of the
original molecule has remained
intact as it served as the template
for the synthesis of a
complementary strand.
• This mode of replication is
described as semi-conservative:
one-half of each new molecule of
DNA is old; one-half new.

Interphase: G1, S and G2 phases
• Lasts much longer than the M phase
• Sometimes referred to as the ‘resting’ phase – this is
UNTRUE as although it doesn’t look like much is
happening, in biochemical terms, this is a very active
period of CELL GROWTH & METABOLISM
– Protein synthesis takes place
– Cytoplasmic organelles are synthesised
– The cell grows and replicates its chromosomes [only
during S phase]

• Interphase is divided into 3 parts:
(1) G1 – First ‘Gap’ phase (During this time the cell is
very active, growing and carrying out metabolic
processes)
(2) S - DNA replication (The 'S' stands for synthesis
as during this phase DNA is synthesized in the
process of replication. Each chromosome becomes
two sister chromatids)
(3) G2 - Second ‘Gap’ phase (In this period
mitochondria and other organelles are divided so
that each daughter cell will have an equal number
of organelles)

Mitosis: the M phase
• Interphase is followed by M Phase which consists of
mitosis and cytokinesis.
• Mitosis is the division of the contents of the nucleus
(PMAT), whilst cytokinesis (CK) refers to the division
of the cytoplasm.
• Cell division involves mitosis and cytokinesis. The
growth of an organism and the replacement of its cells
for tissue repair both depend on mitosis and
cytokinesis.

Control of the Cell Cycle
• A central mechanism is used to assess the status of
the cell as it progresses through the cycle. This
system works through 3 main checkpoints:
• G1 Checkpoint: towards the end of the S phase.Size of the cell is
assessed - if sufficient growth has occurred i.e. cell large enough
for division, then S phase can proceed
• G2 Checkpoint: the success of DNA replication is monitored. If
successful the cell cycle will continue to mitosis
• M Checkpoint: during metaphase prior to anaphase and telophase
triggers exit from from mitosis and cytokinesis and entry into
next G1 phase for daughter cells

Abnormal Cell Division : Cancer cells
• Normal cell development will break down if the control
of cell division, cell growth or cell death fails
• If cell division or cell growth fails, TUMOURS arise
• These can either be benign - don’t cause serious
problems and can be removed by surgery or malignant -
enter the circulation, migrate and proliferate to form
new tumours in new areas of the body. This is called
METASTASIS

Causes of Cancer
• Somatic Cell mutations
• Proliferation genes (proto-oncogenes -> oncogenes)
• Anti-proliferation genes (also known as Tumour-
suppressor genes)

Mitotic Index
• Fraction or percentage of cells in a given sample that
contain condensed chromosomes i.e. the cells are
undergoing mitosis and dividing
http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/mitosis.htm

GLOSSARY
• Checkpoints: Where stop and start signals regulate
the cycle; register internal and external cell signals
which report the state of crucial processes and if the
cycle should proceed.

Chemotherapy
• Chemotherapy is the use of anti-cancer (cytotoxic) drugs to
destroy cancer cells (including leukaemias and lymphomas).
There are over 50 different chemotherapy drugs and some
are given on their own, but often several drugs may be
combined (this is known as combination chemotherapy).
• Chemotherapy may be used alone to treat some types of
cancer. Sometimes it can be used together with other types
of treatment such as surgery, radiotherapy, hormonal
therapy, immunotherapy, or a combination of these.

How do chemotherapy drugs work?
• Chemotherapy drugs interfere with the ability of a cancer cell to divide
and reproduce itself. As the drugs are carried in the blood, they can
reach cancer cells all over the body. The chemotherapy drugs are taken
up by dividing cells, including some normal cells such as those in the lining
of the mouth, the bone marrow (which makes blood cells), the hair
follicles, and the digestive system. Healthy cells can repair the damage
caused by chemotherapy but cancer cells cannot and so they eventually
die.
• Chemotherapy drugs damage cancer cells in different ways. If a
combination of drugs is used, each drug is chosen because of its
different effects. Unfortunately, as the chemotherapy drugs can also
affect some of the normal cells in your body, they can cause unpleasant
side effects. However, damage to the normal cells is usually temporary
and most side effects will disappear once the treatment is over.
• Chemotherapy is carefully planned so that it destroys more and more of
the cancer cells during the course of treatment, but does not destroy
the normal cells and tissues.

Multicellular Organisms
• Multicellular organisms are created from a complex
organization of cooperating cells.
• Some cells provide protection; some give structural
support or assist in locomotion; others offer a means
of transporting nutrients.
• All cells develop and function as part of the organized
system -- the organism -- they make up. There must be
new mechanisms for cell to cell communication and
regulation.
• In humans, there are 1014
cells comprising 200 kinds of
tissues!

Cellular Differentiation
• Each of us originated as a single, simple-looking cell -- a
fertilized egg, or zygote -- so tiny that it can barely be
seen without a microscope. (A human egg cell is about
1/100th of a centimetre in diameter, or a bit smaller
than the width of a human hair.)
• Shortly after fertilization, the zygote begins dividing,
replicating itself again and again. Before long, a
growing mass, or blastula, of dozens, then hundreds,
then thousands of cells called stem cells forms; each
stem cell is only one-fourth to one-tenth the diameter
of the original zygote, but otherwise nearly identical to
it

Cellular Differentiation
• Every nucleus of every cell has the same set of genes.
A heart cell nucleus contains skin cell genes, as well as
the genes that instruct stomach cells how to absorb
nutrients.
• Therefore, for cells to differentiate, certain genes
must somehow be activated, while others remain
inactive.
• Genes instruct each cell how and when to build the
proteins that allow it to create the structures, and
ultimately perform the functions, specific to its type
of cell.

Gene Regulation in Bacteria
• Bacteria adapt to changes in their surroundings by
using regulatory proteins to turn groups of genes on
and off in response to various environmental signals
• The DNA of Escherichia coli is sufficient to encode
about 4000 proteins, but only a fraction of these are
made at any one time. E. coli regulates the expression
of many of its genes according to the food sources
that are available to it.

The lac operon
• The best understood cell system for explaining control
through genetic induction is the lac operon
• Jacob & Monod (1961) - regulation of lactose
metabolism in E.coli
• Composed of 3 segments, or loci of DNA:
1. The REGULATOR - composed of gene that codes for a
repressor protein which can repress the operon.
2. The CONTROL locus - consists of a promotor and the
operator - can start transcription of the structural genes
3. The STRUCTURAL locus - contains structural genes
encoding the enzyme β-galactosidase

The lac operon
In an E. Coli cell growing in the absence of lactose, a
repressor protein binds to the operator, preventing RNA
polymerase from transcribing the lac operon's genes. The
operon is OFF
When the inducer, lactose, is added, it binds to the
repressor and changes the repressor's shape so as to
eliminate binding to the operator. As long as the operator
remains free of the repressor, RNA polymerase that
recognizes the promoter can transcribe the operon's
structural genes into mRNA. The operon is ON

The lac operon
Lactose absent :

The lac operon
Lactose present :

Mammalian Cell Culture
• The ability to grow cells in culture i.e. in the lab, is
essential for biotechnology and research

Applications of cell culture 1
RESEARCH (small scale usage)
• growing bacterial cells for basic gene manipulation
• culturing mammalian cells to observe the effects of
drugs and hormones on the functioning of cells e.g.
cancer studies
• producing new plants

Applications of cell culture 2
BIOTECHNOLOGY (large scale usage)
• agriculture e.g. silage production
• pharmaceuticals e.g. genetically engineered bacteria to
produce insulin
• food production e.g. brewing and baking
• biodegradation e.g. sewage treatment

Conditions needed for cell culture
In order for cells to grow, the conditions must be
just right for each cell type. The cytologist must
therefore consider the following:
•Growth medium
•Type of growth container or fermenter
•Temperature
•pH
•Gas exchange
•Aseptic conditions
•Method for monitoring cell growth
•Safety measures and implications

Aseptic conditions
• To avoid contamination of growth media and
cultures
• All inanimate and living objects, including the
atmosphere carry large numbers of microorganisms.
• A variety of techniques can be used to provide these
conditions:
e.g. sterilisation of all utensils and media using heat.
For example, using an autoclave (steam under pressure,
necessary for bacterial spores)
Growth of pure cultures

Microorganisms
• They are everywhere!
• They highly adaptable to their surrounding environment
• They are relatively easy to culture
• They incredibly diverse and are able to colonise very extreme conditions e.g. salt pans, hydrothermal vents in the ocean floor

Classes of Microorganisms
• There are 2 recognised categories of micro-organism:
1. Unicellular Algae / PHOTOTROPHS: use sunlight to make their own food
1. Bacteria & Yeasts (Fungi) / HETEROTROPHS: need more complex media containing an organic carbon source and other compounds e.g. amino acids

Culture & Uses
• Food industry - cheese production, baking, wine & beer
• Chemical production e.g. acetone
• Bases of food chains
• Commensal bacteria in digestive tract
• Production of therapeutic compounds e.g. insulin
[See Scholar: Batch & Continuous culture]

Microbial Growth Culture Requirements
• A few litres can be made in the lab
• Thousands of litres can be made industrially
• Micro-organisms are grown in a medium that supplies
them with all nutrients necessary for growth.
• This depends on …
– the type of cell
– the final purpose of the cell
– the by-products

Microbial Growth Culture
Requirements
• Important factors that must always be considered are:
– the nutrient media
– temperature
– pH
– gaseous environment
– light
Unicellular algae, bacteria & yeast can be grown as batch
cultures - no dilution is needed until max. density is reached.
Growth can be limited by nutrient availability i.e. at the end of
exponential growth

Nutrient Media
• Chosen to imitate an organism’s natural environment
• Generally supplies all the essential nutrients
• A medium is classed as any solid or liquid preparation
specifically for growth, storage or transport of micro-
organisms
• Must be at the correct pH and the correct gaseous
concentration for the organisms to grow

Nutrient Media
• There are 2 types of media commonly used:
1.1. Complex mediaComplex media - this has one or more crude
sources of nutrients and their exact chemical
composition and components are not known. Generally
used for routine cultures
2.2. Defined mediaDefined media - otherwise known as
synthetic media containing chemically known
compounds and components which are in a relatively
pure form
REMEMBER: all media must be STERILESTERILE before use !!!

Mammalian Cell Culture
• Many animal cells and tissues can be removed from an
organism and cultured artifically. This allows the cell’s
activities to be investigated e.g. control of the cell
cycle

The process of culturing Mammalian Cells
Once the cells are obtained from animal tissues or
other cell lines they are placed in a flat culture vessel
that lies on its side
The cells stick or adhere to the inside of the vessel as
they grow in the medium
Most animal cells are ‘ANCHORAGE-DEPENDENT’ i.e. they
need something to hold on to
These cells usually form a monolayer that will eventually
cover the entire surface of the medium

At this point, called confluence, it is necessary to
subculture the cells into a fresh medium
N.B.N.B. Cells that are associated with body fluids such
as blood cells are NON-ANCHORAGE DEPENDENT and can
be grown in suspension. Again, it is necessary to
regularly subculture the cells into fresh medium
N.B.N.B. All media and culture vessels are STERILISED to
prevent the growth of micro-organisms

Mammalian Cell Growth Media
• Contains …
- mixture of glucose, amino acids, salts, water
and antibiotics
- sometimes BASIC GROWTH SERUM is added
[This is animal serum prepared from blood and
contains additional factors e.g. Platelet Derived
Growth Factor,which enhances growth, 5-10% added or Fetal
Bovine Serum (FBS) ]
- pH indicator e.g. phenol red: this shows
changes in pH due to waste production
(pH decreases  red to yellow)
* Finally, the media must be incubated at the appropriate
temperature for the chosen cells e.g. human cells - 37o
c *

Categories Of Mammalian Cell
Cultures
• There are 2 categories of animal cell cultures:
(1)Primary cultures:
• These cells are taken directly from fresh
tissue.
• The disadvantage is that the cells have a
limited lifespan; the cells only divide so many times
in culture, so therefore long term culturing is
difficult

Process of Cell Collection
• The cells are treated with a proteolytic enzyme e.g.
trypsin, to separate out the fragments into single cells.
• The advantage of this process is that cells can be
collected and cloned.
• This is useful to isolate a mutant cell line i.e. deriving
secondary cell cultures otherwise known as ...

(2) Continuous Cell Lines
• These cells have an acquired capacity for infinite
growth and division [they are immortal]
• They are derived from tumours or the cells have been
transplanted [neo-plastic - produce cancer if
transplanted into animals] so they have lost their
sensitivity to factors associated with growth control.
• Generally, these cells will lose their anchorage
dependence facility and so are often easier to culture

Continuous Cell Lines
• The advantage of using continuous cell lines is that
they can be cloned.
• This allows easy :
– isolation of mutant cells
– investigation of cell growth
– production of hybrid cells in biotechnology
This routine procedure is used to produce important
pharmaceuticals e.g. vaccines and hormones

Bacterial & Fungal Cultures
• Much easier to grow than mammalian cells !
• Bacteria and Fungi require much simpler growth media
requirements and culture conditions compared to
animal cells.
[See previous notes]

Plant Tissue Culture
• One major problem in plant breeding is that crosses
can only be made between closely related parental
types. This makes it very difficult to introduce new
genes into a plant species.
• The solution to this problem is PROTOPLAST FUSIONPROTOPLAST FUSION -
protoplasts of different plants are mixed and fused
together. These form a binucleate cell containing a
nucleus from both parental types
ProtoplastProtoplast = actively metabolising part of cell minus
cell wall [cell wall digested by enzymes]

Process of Plant Cell Culture
1. Plant cells treated with cellulase & pectinase to remove the cell
wall which is composed of cellulose, pectin and small amount of
hemicellulose. These enzymes only break down the cell wall leaving
the plasma membrane intact
2. The cells are then incubated with a mineral salt solution containing
mannitol for several hours. This sugar exerts osmotic pressure
causing PLASMOLYSIS leading to easier digestion
3. In order for the protoplasts to grow they must be put in a suitable
medium to encourage cell wall growth

4. EXPLANTSEXPLANTS (small pieces of young growing plant tissue e.g. root,
shoot, bud or leaf) can be taken and grown in a suitable medium
containing plant growth regulators (growth hormones e.g. auxins
and cytokinins whuch cause tissue differentiation).
Cell proliferation produces a CALLUSCALLUS (a mass of dividing,
undifferentiated cells)
5. With continued sub-culturing and changing the balance of growth
regulators, the new roots and shoots can be planted out to
regenerate a complete plant !

Totipotency
• All plant cells are totipotenttotipotent - they each have the
ability to express the full genetic potential of that
plant

STRUCTURE & FUNCTION OF
CELL COMPONENTS

Introduction
• Living systems are composed of a limited number of
elements namely…
CARBON, HYDROGEN, OXYGEN, NITROGEN,CARBON, HYDROGEN, OXYGEN, NITROGEN,
PHOSPHORUS & SULPHURPHOSPHORUS & SULPHUR
• The carbon atom is of central biological importance as it
can form 4 covalent bonds with other atoms
• This allows a variety of complex molecules to be constructed
• Many functional chemical groups are also associated with biological molecules as they are important in biological systems

Polymers
• Many biologically important
molecules are polymers
composed of monomers linked
together
• Two monomers are joined
together by removing water
molecules. This is called a
CONDENSATION reaction or
DEHYDRATION synthesis
• This can be reversed by
adding (back) water ->
HYDROLYSIS
• This is an important feature
of cell metabolism
Dehydration
Hydrolysis

• Making and breaking chemical bonds involves ENERGYENERGY
• Synthesising more complex structures REQUIRES
energy. These are called ANABOLIC or
BIOSYNTHETIC reactions
• If there is little overall change in energy, the
reactions are reversible
• Cell metabolism is tightly controlled to avoid energy
chaos

Carbohydrates
• Composed of CARBON, HYDROGEN & OXYGEN
MONOSACCHARIDES ‘Single Sugars’ e.g. glucose, fructose
- General formula (CH2O)n
- classified by number of carbons they have
n = 3 TRIOSE
n = 5 PENTOSE
n = 6 HEXOSE
- structure can vary greatly depending on the
number of C atoms and the arrangement of H
and O atoms

Glucose (C6H12O6)
• Hexose sugar
• Can exist in different forms
depending on the position of the
carbonyl group (C=O) on the terminal
carbon
• Variations of C6H12O6 are called
isomers
• If OH group on C5 projects to the
right = D Form (most common)
on left = L Form
D-GLUCOSE = straight chain form
of glucose (C6H12O6)

• In solution, glucose adopts a cyclic form where C1 and C5
are linked by an oxygen atom giving a ring structure
(see diagram)
• Depending on the position of the -OH group on C1
whether:
– (α) alpha - below C1
– (β) beta - above C1
• In solution the equilibrium proportions of the three
forms are approximately 38% α to 62% β to 0.02%
straight chain glucose at any given time

The Glycosidic Bond
• 2 monomers (monosaccharides) can be linked by
DEHYDRATION SYNTHESIS or the CONDENSATION
REACTION, to give a disaccharide
• The carbohydrate’s name is defined by the component
monomers and the way the bond is arranged
• Common disaccharides are :
SUCROSE = Glucose + Fructose
LACTOSE = Glucose + Galactose
ANIMATIONANIMATION

Polysaccharides
• Long chains of simple sugars e.g. starch, glycogen and
cellulose
• If the repeating monomers are the same, they form a homopolymer. If
they are different they form a heteropolymer
• Polysaccharides are insoluble in water and so make
ideal storage compounds
• The following three polysaccharides are all homopolymers of glucose but
they have different functions and properties depending on their structure

1. Starch
• Found in plants
• Helical arrangement of glucose
• Storage polysaccharide of
energy
• Can be easily hydrolysed to
release monomers of glucose for
energy
• Starch test: turns iodine from
dark brown to blue/black

2. Glycogen
• Storage compound in animals,
generally found in the liver
• Polymer of glucose linked by α
1-6 bonds and α 1-4 bonds
• Short term energy store
• Plays a role in homeostatic
control of blood sugar level
• Remains dark brown with
iodine

3. Cellulose
• Storage compound in plants
• Parallel chain arrangement linked by β 1-4
glycosidic bonds and hydrogen bonding
between parallel chains
• Doesn’t stain with iodine
• Very tough arrangement of fibres due to
structural arrangement
• most abundant organic material on Earth
• Most animals lack cellulase, the enzyme
needed to breakdown the component
monomers

4. Chitin
– A homopolysaccharide similar to cellulose in
structure. Component of many insect exoskeletons -
very strong and rigid; also resistant to chemicals.
5. Glycosaminoglycans
– A heteropolymer found in skin and connective tissue
of vertebrates

Summary of Carbohydrate Functions
• Immediate respiratory substrate e.g. glucose
• Energy stores e.g. glycogen in mammals, starch in plants
• Structural components e.g. cellulose in plant cell walls, chitin in insect
exoskeleton, pentose sugars (ribose & deoxyribose in RNA & DNA)
• Metabolites i.e. intermediates in biochemical pathways
• Cell to cell attachment molecules e.g. glycoproteins or glycolipids on the
plasma membrane
• Transport e.g. sucrose in plant phloem tissue

Structure & Function of Lipids
• Lipids are organic compounds found in every type of plant
and animal cell.
• They contain the elements CARBON, HYDROGENCARBON, HYDROGEN and
OXYGENOXYGEN [but less O2 than in carbohydrates]
• All lipids are INSOLUBLE in WATER
• Lipids have many important functions:
– In cell membrane structure - Mechanical Protection
– Hormones - Electrical Insulation of Nerves
– Energy storage molecules - Waterproofing & Buoyancy
– Thermal Insulation

• FATS: Solid at room temperature
– SATURATED FATSSATURATED FATS:: all available bonds are occupied by
Hydrogen
Most animal fats are saturated e.g. butter, lard
• OILS: Liquid at room temperature
– UNSATURATED FATS:UNSATURATED FATS: contain C-C double bonds in the
molecule therefore kinks are introduced.
Oils tend to be more available in plants e.g. sunflower
oil, olive oil

Type of Lipids
• 3 types of lipids which are important to cells:
1.Triglycerides
• Most common type of lipid
• 3 fatty acids and a glycerol molecule are linked by an ester bond
formed during dehydration synthesis
2. Phospholipids
• Same as triglycerides except one of the fatty acids molecules is
replaced by a phosphate group (PO4
3
-)
• The phosphate group is polar and so is attracted to water –
therefore the phospholipid has two distinct ‘ends’
• A hydrophilic end (‘water loving’) that dissolves in water and a
hydrophobic end (‘water hating’) that is repelled by water
3. Steroids
• Very different structure – 4 carbon rings with variety of different
side chains

Triglycerides cont.
• The properties of triglycerides are determined
by their constituent fatty acids
• DEHYDRATION SYNTHESISDEHYDRATION SYNTHESIS occurs between the
hydroxyl group of the glycerol molecule and
the carboxyl groups of the fatty acid molecule
producing an ester
• Main function = ENERGY STORE e.g. camel hump
• The form in which fatty acids are transported
round the body and stored is adipose tissue
ANIMATION

Phospholipids
• Similar to triglycerides but one fatty acid is
replaced by a phosphate group which often
has other groups attached
• Usually one fatty acid is saturated and one is
unsaturated. Most common phospholipid in
animal tissue is PHOSPHATIDYLCHOLINEPHOSPHATIDYLCHOLINE
•The phospholipid has two distinctive
ends:
–HYDROPHILIC HEADHYDROPHILIC HEAD that dissolves in water
–HYDROPHOBIC TAILHYDROPHOBIC TAIL that repels water
This property causes phospholipids to spontaneously form bilayers

Functions of Phospholipids
• Essential components of cells and
organelle membranes
• Components of lung surfactants

STRUCTURE & FUNCTIONS OF
PROTEINS
• Proteins are essential in biological systems as controlscontrols
e.g enzymes and structural elementsstructural elements e.g. cytoskeleton
• Proteins are heteropolymers as they are made up of
different amino acids (20 different types)
• The type and order of amino acids determines the
structure and function of proteins allowing them to
carry out many different roles

Amino Acids
• Amino acids are characterised by the amino group
(NH2) and the carboxylic acid (COOH)
• These are attached to a central carbon atom which
also carries a hydrogen
• The side chains are variable, the ‘R’ group can be joined
here

• At neutral pH, amino acids exist in ionised forms. Once
joined, the charges on amino acids disappear.
R GROUP
• This gives the amino acid it’s unique chemical properties
and specific shape.
• The R group can be classified as acidic, basic, uncharged
polar or non-polar

Types of Amino Acids
Amino Acid
Class
Name Abbreviations R-Group
Acidic Aspartic Acid Asp/D - CH2COOH
Basic Lysine Lys/K - (CH2)4NH4
Uncharged
Polar
Serine Ser/S - CH2OH
Non-polar Glycine Gly/G - H

The Peptide Bond
• Proteins are made by joining amino acids together by
an amide linkage / peptide bond
• A chain of amino acids is called a polypeptide
• The peptide bond is formed by DEHYDRATIONDEHYDRATION
SYNTHESISSYNTHESIS or a condensation reaction between the
carboxyl group of one amino acid and the amine group
of the next amino acid
• Amino acids joined in this way are called residues

The Peptide Bond
• The Peptide bond is very
strong
• C-N bond is planar (flat) so
peptide bond allows NO
rotation
• The single bonds either side
DO allow rotation of the
residues, so polypeptide
chains are flexible
ANIMATION

Protein Structure
• Chemical bonding is critical in determining a protein’s
shape and the different types of bonds are important
for different levels of protein structure
PEPTIDE BOND = COVALENT BOND = VERY STRONGPEPTIDE BOND = COVALENT BOND = VERY STRONG
• In higher order protein structures, weaker
interactions are important too.These include:
– Non-covalent bonds
– Hydrogen Bonds
– Ionic bonds
– Van der Waals interactions
– Hydrophobic interactions between R groups

Primary Structure (1o
)
• Primary structure refers to the "linear" sequence of amino
acids.
• The amino end or N terminus is positioned to the left.
The carboxyl end or C terminus is positioned to the
right
N
C
Generally 3 or 1 letter
abbreviations are used
to denote amino acids
when primary structures
are drawn

Secondary Structure (2o
)
• Secondary structure is "local" ordered structure
brought about via hydrogen bonding mainly within the
peptide backbone
• A single polypeptide many contain several secondary
structures
• The most common secondary structure elements in
proteins are the alpha (α) helix and the beta (β)
sheet (sometime called b pleated sheet)

Tertiary Structure (3o
)
• This describes the way in which the polypeptide folds to
give the final structure of the protein.
• The 3o
structure is determined by hydrophobic
interactions which place the amino acids non-polar R
groups towards the centre of the molecule
• In many proteins an additional important type of bond is
the disulphide bond. This bond forms between sulphydryl
(SH) groups on cysteine residues; so may be formed
between 2 different polypeptides or within the
polypeptide itself.
• Within any tertiary structure, parts of the amino acid
sequence may adopt an α-helix, β-sheet or more complex β
sheet arrangements e.g. myoglobin

• The ion group is a prosthetic group – a non-protein
group associated with a folded protein
• If the attached group is :
– CARBOHYDRATE = Glycoprotein
– LIPID = Lipoprotein
– NUCLEIC ACID = Nucleoprotein
These are known as
conjugated proteins

• As proteins have a relatively stable structure in a
cellular environment, it is remarkable that the forces
that hold them together can be easily disrupted if the
chemical environment changes or the sequence of
amino acids is changed

• Alpha Helix
• The polypeptide chain is coiled into a right handed
helix by Hydrogen bonding (stabilises the helix)
between the NH group of the peptide and the C=O of
the peptide bond, four residues away from it

Beta sheet
• The polypeptide chains are linked together in a side by
side configuration by hydrogen bonding. Beta sheets
can be either parallel or anti-parallel depending on the
orientation of the constituent parts

Quaternary Structure (4o
)
• Proteins that are composed of 2 or more polypeptide
sub-units

Nucleic Acids [revise Higher notes]
• DNADNA and RNARNA are information carrying molecules
– DNADNA: info storage & transmission
– RNARNA: protein synthesis
• Simple chemical structure based on a SUGARSUGAR
PHOSPHATE BACKBONEPHOSPHATE BACKBONE
• Coding part made of 4 nitrogenous bases which arrange
themselves in pairs
• This enables a massive variety and diversity of
proteins to be built
[Diagram of Nas/Nucleotides]

Nucleotides
• Monomer of nucleic acid
• Consists of 3 main parts :
– a PENTOSEPENTOSE sugar (deoxyribose/ribose)
– a PHOSPHATEPHOSPHATE group (PO4
2-
)
– a nitrogenous base (PURINEPURINE or a PYRIMIDINEPYRIMIDINE)
PURINE PYRIMIDINE
double or fused ring structure single ring structure
ADENINE, GUANINE
CYTOSINE, THYMINE &
URACIL (only found in RNA)
N.B. Base Pairing: A always bonds with T (or U), G with C

Phosphodiester Bond
• Chains of nucleotides (polynucleotides) formed by
DEHYDRATION SYNTHESISDEHYDRATION SYNTHESIS reaction between the
phosphate group of one nucleotide and the hydroxyl
group on the sugar of another
• This bonding gives polynucleotides a defined polarity
reflecting the component nucleotides
[Diagram of Phosphodiester bond]

Polynucleotides & Nucleic Acid
Function
• Polynucleotide chains provide the structural and
functional basis for the encoding and decoding of
genetic information.
• The sugar phosphate backbone carries a sequence of
bases that makes up the genetic code as a series of
triplet codons
• Complementary base pairing holds the key to copying
genetic information in the processes of DNA
replication and transcription
• The bases fit together A-T(U) and G-C are joined
together by HYDROGEN BONDINGHYDROGEN BONDING
[Base pairing diagram][Base pairing diagram]

DNA
• A double stranded helix composed of
two polynucleotide chains that run in
opposite directions (anti-parallel)
• The bases fit across the right-handed
helix; one purine pairing with its
complementary pyrimidine
• The helix is the only shape that
accommodates the purine-pyrimidine
base pair and maintains stable
hygrogen bonds

RNA
• 3 types of RNA which are SINGLE strandedSINGLE stranded but can
fold to give 3D shapes or conformations:
• mRNAmRNA - contains information transcribed from a DNA
molecule and transports it to a ribosome
• tRNAtRNA - collects amino acids and transports them to a ribosome to be fitted
according to the messenger RNA code
• rRNArRNA (ribosomal RNA) - provides a major structural
support component of the ribosome

Polymerase enzymes
• A polymerase is an enzyme whose central function is
associated with polymers of nucleic acids such as RNA
and DNA
• These are necessary for the following processes:
1) DNA REPLICATION:DNA REPLICATION: enables a complete
copy of the genome to be passed on
to each daughter cell during mitosis
2) TRANSCRIPTION OF DNA into RNA:TRANSCRIPTION OF DNA into RNA: :
mechanism by which genes are
expressed DNA polymerase

DNA Ligase
• This enzyme forms phosphodiester bonds which are
used to join DNA molecules or fragments together to
produce recombinant DNA (recDNA)
Both polymerases,ligases and restriction endonucleases
(cut DNA) are important components of a genetic
engineer’s ‘toolkit’. They are used to manipulate
DNA

Cell Membranes
• The cell membrane/plasma membrane represents the
barrier that separates the cell’s contents from the
surrounding environment and controls what moves in
and out
• In eukaryotic cells, membranes are also used to
generate compartments within the cell, each with a
specialised function e.g. golgi apparatus, endoplasmic
reticulum, lysosomes etc

Membrane functions
• Provides selectively permeable barriers
• Compartmentalisation
• Localises reactions in the cell
• Transport of solutes often against the concentration
gradient (active transport)
• Signal transduction – receptor proteins on the membrane
surface recognise and respond to different stimulating
molecules, enabling specific responses to be generated
within the cell
• Cell to cell recognition – the external surface of the
membrane is important as it represents the cell’s
biochemical “personality”. In multicellular organisms this
allows cells to recognise each other as similar or different,
which is necessary for the correct association of cells
during development.

Membrane Structure
• The basic composition and structure of the plasma
membrane is the same as that of the membranes that
surround organelles and other subcellular
compartments.
• The foundation is a phospholipid bilayer – polar
hydrophilic heads on the outer surface and
hydrophobic non-polar fatty acid tails form the inner
surface. The membrane as a whole is often described
as a fluid mosaic – a two-dimensional fluid of freely
diffusing lipids, dotted or embedded with proteins
which may function as channels or transporters across
the membrane, or as receptors.

• The idea that membranes were composed of
phospholipids was first put forward in 1925. The
currently accepted model for membrane structure was
proposed by S.J. Singer (1971) as a lipid protein model
and extended to include the fluid character in a
publication with G.L. Nicolson in "Science" (1972)
• The fluid mosaic model has 2 components, lipids and
proteins. The lipids form the matrix bilayer of the
membrane and the proteins carry out all of its
functions
• The membrane is not a static rigid structure, but a
dynamic arrangement of lipids and proteins that drift
laterally within it.

Types of Membrane Proteins
• Proteins make up approximately 50% of the mass of the plasma
membrane and can be classified into different groups depending
on their arrangement in the membranearrangement in the membrane and/or their functionfunction
• Proteins may be embedded in the lipid bilayer or attached to the
surface
• The embedded or INTRINSIC proteins may be transmembrane
proteins (span the bilayer) or they may be linked to lipids on one
side of the bilayer only
• The peripheral or EXTRINSIC proteins are loosely attached to
the membrane by ionic association with other proteins
• Glycoproteins have carbohydrates attached to their extracellular
domains.

Functions of Membrane Proteins
• The main functions of these membrane proteins are as
follows:
–Transport
–Cell recognition
–Receptor sites
–Enzymes
–Intracellular Junctions

1) TRANSPORT PROTEINS
• Transport non-diffusable substances across the membrane. May
be either:
(a) Channel proteins – provide a ‘pore’ across the membrane
through which molecules (usually small and charged) can diffuse
(b) Carrier proteins – these are more specific with binding
sites for only one solute
• Both these proteins permit passive transport (with a
concentration gradient this is called facilitated diffusion)
• To transport molecules against the concentration gradient, special
types of the carrier proteins are needed. These harness energy to
drive the transport process during active transport e.g. sodium-
potassium pump

2) CELL RECOGNITION PROTEINS
• Usually glycoproteins
• The carbohydrate chain of the glycoprotein projects
out of the cell enabling cell to cell recognition and
serving as a cell “fingerprint”
• Therefore, the immune system can recognise it’s own
cells and organs e.g. ABO blood group antigens:
– A = glycoprotein antigen A
– O = no glycoprotein antigens

3) RECEPTOR PROTEINS
• These have a specific conformation (shape) that allows
binding of a particular molecule (the ligand)
• The binding of the ligand will then trigger a response in
the cell

4) ENZYMES
• A protein that catalyses a specific reaction
• Some receptor proteins have enzymatic activity; the
cytoplasmic portion of the protein catalyses a reaction
in response to binding a ligand

5) INTRACELLULAR JUNCTIONS
• Interactions between the plasma membranes of
different cells is a frequent occurrence and takes
place at cell junctions e.g.
-> In PLANTSPLANTS
• PLASMODESMATA – although each plant cell is
encased in a boxlike cell wall, fine strands of
cytoplasm, called plasmodesmata, extend through
pores in the cell wall connecting the cytoplasm of
each cell with that of its neighbors allowing direct
exchange of materials

– In ANIMALSANIMALS, there are 3 types…
• Spot desmosome – dense protein deposits that hold
adjacent cells together by rivets. Mechanical strength is
provided by the intracellular filaments passing from one
desmosome to another
• Tight junction – adjacent membrane proteins are bonded
together preventing movement of materials in the space
between the cells e.g. between epithelial cells lining the small
intestine
• Gap junction – doughnut shaped proteins from each cell
joined together to form tiny channels allowing the passage of
small molecules such as ions, amino acids and sugars

The Cytoskeleton
• The eukaryotic cell is a 3D
structure. It has a
cytoskeleton anchored to
proteins in the plasma
membrane
• These proteins both maintain
shape and allow movement
• The cytoskeleton is a dynamic
structure, as the
microfilaments and
microtubules can
depolymerise and
repolymerise very easily
MICROFILAMENTS
MICROTUBULES
INTERMEDIATE
FILAMENTS

The Cytoskeleton
• The cytoskeleton is made up of 3 components, in order
of increasing diameter. They are …
1) Actin filaments/microfilaments
2) Intermediate filaments
3) Microtubules

1) Microfilaments
• These are composed of actin (protein)
• They are arranged as 2 strands of
protein molecules twisted together to
give a rope-like structure about 7nm in
diameter
• These are present throughout the cell
but are most highly concentrated just
inside the plasma membrane
• They are important in all cell movementcell movement
and contractioncontraction Actin fibres in a cell stained with
a fluorescent strain specific for
actin

2) Intermediate Filaments
• These are about 10nm in diameter
and are composed of tough fibrous
protein strands twisted together
• They are very stable structures in
the cell and provide mechanicalmechanical
strengthstrength to animal cells which lack
the strong cell walls of plants
• Intermediate filaments can be
anchored between the membrane to
provide extra support
The nucleus in epithelial cells is held
within the cell by a basketlike
network of intermediate filaments
made of keratins which have been
stained here using a fluorescent stain

3) Microtubules
• These are hollow tubes (like straws)
composed of tubulin protein (a globular
protein)
• The tubulin protein subunits of microtubules
associate in a cylindrical arrangement to
generate the final microtubule - a relatively
rigid structure
• Microtubules only form around a
centrosome (organising centre)
• The centrosome provides a “nucleus” from
which the microtubules form. These are
important in cell division as part of the
spindle fibre network and can move
components within the cell
Microtubules growing in
vitro from an isolated
centrosome

Functions
But the primary importance of the cytoskeleton is in cellcell
motilitymotility. The cytoskeleton extends throughout the
cytoplasm and determines the internal movement of cell
organelles, as well as cell locomotion and muscle fibre
contraction
All of these
components give
mechanical supportmechanical support
and shapeshape to the cell

MOLECULAR INTERACTIONS IN
CELL EVENTS

Molecular Interactions In Cell
Events
• CATALYSISCATALYSIS:
- The vast number of coordinated and complex
biochemical reactions that occur in an organism is
summarised as the cell METABOLISMMETABOLISM
- The reactions are in ordered pathways, controlled at
each stage by ENZYMESENZYMES
- Through these metabolic pathways, the cells are able
to transform energy, breakdown macromolecules and
synthesise new organic molecules needed for life

Anabolic Reactions
• Uses energy to
SYNTHESISESYNTHESISE large
molecules from smaller
ones e.g.
Amino Acids->Proteins
• Also known as endothermicendothermic
reactions
ENDOTHERMIC REACTION

Catabolic Reactions
• These release energy
through the BREAKDOWNBREAKDOWN
of large molecules into
smaller units e.g.
Cellular Respiration:
ATP -> ADP + Pi
• Also known as exothermicexothermic
reactions
EXOTHERMIC REACTION

Naming Enzymes
• Enzymes are commonly named by adding a suffix "-ase" to
the root name of the substrate molecule it is acting upon.
For example, Lipase catalyzes the hydrolysis of a lipid
triglyceride. Sucrase catalyzes the hydrolysis of sucrose
into glucose and fructose
• A few enzymes discovered before this naming system was
devised are known by common names e.g. pepsin, trypsin, and
chymotrypsin which catalyse the hydrolysis of proteins
• Enzymes are also given a standard reference number
(European Commission Number) to help characterise the
1500 or so enzymes

ENZYMES
These catalyse a transfer of a phosphate group onto a
molecule such as a carbohydrate or protein
Kinases
To hydrolyse ATP. Many proteins have an ATPase activity
which is essential for their functionATPases
To hydrolyse phosphodiester bondsNucleases
To hydrolyse peptide bonds to breakdown proteins ->
amino acids
Proteases
FUNCTIONNAME

Form & Function of Enzymes
• Enzymes work by bringing about substrate(s) of a
reaction close together in an active siteactive site so that bond
breakage or formation occurs at atomic level
• This is often facilitated (helped) by specific chemical
effects such as the transfer of proteins or the
alteration of charge distribution around the target
atoms
• The substrate and enzyme must fit together veryvery
preciselyprecisely

The Catalytic Cycle
• A cycle of events that describes an enzyme
combining with a substrate, remaining unchanged by
the reaction and being available at the end of the
reaction to combine with another substrate molecule

The Catalytic Cycle of Sucrase
Sucrase catalyses the hydrolysis of
sucrose into it’s component
monosaccharides, GLUCOSE &GLUCOSE &
FRUCTOSEFRUCTOSE
1) At the start of the cycle, enzyme (E)
and substrate (S) are available
2) The molecular interaction of enzyme
and substrate at the active site forms
the enzyme:substrate complex (ES)
3) Catalysis occurs, forming the
enzyme:product complex (EP)
4) Products are released, leaving the
enzyme free for the next substrate
molecule
E
S
ES
EP

Model for Enzyme Action
• A common model for enzyme action is the lock and keylock and key
hypothesishypothesis
• However, this model is a little misleading in that it
tends to give the impression that enzymes are rigid
structures, whereas in fact, they are quite flexible and
can alter their conformation in response to the binding
of other molecules
• The currently accepted model for enzyme action is the
INDUCED FIT MODELINDUCED FIT MODEL, in which conformational changes
to the protein occur on binding of a substrate

The Induced Fit Model
• The enzyme, HEXOKINASEHEXOKINASE, catalyses the transfer of a
phosphate from ATP onto glucose
• The active site and the two domains of the single
polypeptide chain are clearly visible in the view of the
backbone of the molecule
• Think of the protein about to close around the
substrate in the active site similar to the way your
hand would close around a door handle
• The effect of this is that glucose fits the active site
more closely, and the binding of ATP is also enhanced
[see diagram of ‘The catalytic cycle of hexokinase]

Control of Enzyme Activity
• The activity of enzymes must be reguated in some way
to avoid metabolic chaos
• Regulation can be achieved through a number of
different mechanisms
• A major influence is the NUMBER OF ENZYMES MOLECULES
in the cell, which is controlled at the level of gene
expression
• COMPARTMENTALISATION also enables the cell to keep
sets of enzymes together and away from other
enzymes
• TEMPERATURE & pH also affect enzyme activity
• Many enzymes also require CO-FACTORS to function

• However, the most effective way of enabling a fine
control of enzyme activity is to alter the shape of the
enzyme itself, and thus cause a change in its catalytic
efficiency
• Examples of this type of metabolic control include
INHIBITORS,INHIBITORS, ALLOSTERIC EFFECTORSALLOSTERIC EFFECTORS,, COVALENTCOVALENT
MODIFICATIONMODIFICATION and END-PRODUCT INHIBITION

Inhibitors
• Enzymes reaction rates can be changed by competitive
inhibition and non-competitive inhibition
• Inhibitors can be either competitive or non-
competitive

COMPETITIVECOMPETITIVE inhibitors compete for the active site of the
enzyme, thus reducing its effectiveness
– competitive inhibitors are usually similar in structure to
the substrate and the enzyme is ‘fooled’ into accepting the
inhibitor, which blocks the active site

E.G: An example for competitive inhibition is
the enzyme succinate dehydrogenase by
malonate. Succinate dehydrogenase catalyses
the oxidation of succinate to fumarate.

NON-COMPETITIVENON-COMPETITIVE inhibitors bind at a different location and
change the conformation of the enzyme, thus altering the
shape of the active site and again reducing the catalytic
efficiency

• Inhibition can either be reversible or non-
reversible depending on how the inhibitor binds to
the enzyme
• Some inhibitors bind irreversibly with the enzyme
molecules, inhibiting the catalytic activities
permanently. The enzymatic reactions will stop
sooner or later and are not affected by an increase
in substrate concentration. These are irreversibleirreversible
inhibitorsinhibitors.
• Examples are heavy metal ions including silver,
mercury and lead ions.

Allosteric Enzymes
• These are enzymes that ‘change shape’ in response to
the binding of a regulating molecule (often called a
modulator or effector)
• Allosteric modulators can be either positive or
negative effectors of enzyme activity
• They function by binding to allosteric sites that are
distinct from the active site of the enzyme
• Non-competitive inhibition is a form of allosteric
regulation

• In multi-subunit enzymes, the structure is more
complex, and the enzyme often exists in 2 different
conformational states:
– ACTIVE and INACTIVE
• These can be stabilised by binding the modulator
– Positive modulators stabilise the active form of the
enzyme
– Negative modulators stabilise the inactive form
• In addition to these modulators changing the activity of
allosteric enzymes, sometimes the binding of the
substrate itself to one active site enhances binding at
the other active sites. This is known as COOPERATIVITY

Covalent Modification
• Covalent modification of enzymes is another strategy
used widely in metabolic regulation
• One of the most common modifications is the addition
of a PHOSPHATEPHOSPHATE group, which can alter the shape of a
protein by attracting positively charged R-groups
[phosphates carry 2 negative charges on the 2 single-bonded O
atoms]
• PROTEINPROTEIN KINASESKINASES add phosphate groups and
PHOSPHATASESPHOSPHATASES remove them, thus the effect can be
REVERSEDREVERSED
• Some proteins are activated by phosphorylation,
others are inactivated

• An example of phosphorylation activating an enzyme is
the skeletal muscle enzyme GLYCOGEN PHOSPHORYLASEGLYCOGEN PHOSPHORYLASE
• This enzyme releases glucose molecules from glycogen
when heavy demands are placed on muscle tissue
• This process is highly regulated. Traffic of sugar into
and out of storage in glycogen is used to control the
level of glucose in the blood, so glycogen
phosphorylase must be activated when sugar is needed
and quickly deactivated when glucose is plentiful

• Glycogen phosphorylase is present as an inactive non-
phosphorylated form which is converted to the active
phosphorylated form by the addition of a phosphate
group to a serine residue in the protein by the enzyme
PHOSPHORYLASE KINASEPHOSPHORYLASE KINASE
• When the demand for glucose drops, PHOSPHORYLASEPHOSPHORYLASE
PHOSPHATASEPHOSPHATASE removes the phosphate group and
inactivates the enzyme
However … glycogen phosphorylase is also regulated by an
allosteric effect !

• Glucose and ATP act as negative modulators and AMP
(adenine monophosphate) acts as a positive modulator –
also causing the enzyme to shift to the active
conformation
• This is useful, because AMP is a product of ATP
breakdown and will be more plentiful when energy
levels are low and more glucose is needed
• A further complication is that there is a hormonal
control mechanism by adrenaline and glucagon

Proteolytic Cleavage
• Another form of control by a covalent activating
mechanism is proteolytic cleavage as found in the
enzyme TRYPSINTRYPSIN
• Trypsin is synthesised in the pancreas, but not in its
active form as it would digest the pancreatic tissue
• Therefore it is synthesised as a slightly longer protein
called TRYPSINOGENTRYPSINOGEN, which is inactive
• Activation occurs when trypsinogen is cleaved by a
protease in the duodenum
• Once active, trypsin can activate more trypsinogen
molecules, resulting in an autocatalytic cascade that
produces a large number of active trypsin molecules very
rapidly

End-Product Inhibition
• Metabolism is organised as a series of metabolic
pathways, and control of these pathways is an
important feature of cell biochemistry
• One way in which control can be exercised is END-END-
PRODUCT INHIBITIONPRODUCT INHIBITION
• End-product inhibition is energetically efficient as it
avoids the excessive (and wasteful) production of the
intermediates of a pathway
• This is a form of NEGATIVE FEEDBACKNEGATIVE FEEDBACK

For example, in the production of the amino acid isoleucine in bacteria,
the initial substrate is threonine which is converted by five intermediate
steps to isoleucine. As isoleucine begins to accumulate, it binds to an
allosteric site of the first enzyme in the pathway thereby slowing down its
own production. In this way, the cell does not produce any more
isoleucine than is necessary.

The Sodium-Potassium Pump
(Na+/K+ - ATPase)
• A specific case of active transport
• This is one of the best examples of active transport in
animal cells
• This pump transports Na+ ions out of the cell and K+ ions
into the cell. Thus keeping the intracellular concentration
of Na low compared to outside, and the intracellular
concentration of K high
• The pump is driven by hydrolysis of ATP
• It uses about 30% of the energy available to any one
animal cell!
• The pump is a transmembrane carrier proteintransmembrane carrier protein made up of
4 subunits (2 large and 2 small)

• Structure: has 3 binding sites for sodium ions, 2
binding sites for potassium ions and a phosphorylation
site to accept a phosphate from ATP
• 2 different conformations of protein are possible.
This is controlled by the phosphorylation state of the
protein
• Hydrolysis of one ATP molecule fuels the export of 3
Na+ ions and the import of 2 K+ ions
• Can work as fast as 300 Na+ ions per second if
required!
[Sodium-Potassium Pump Diagram]
Sodium Potassium Pump Animation

Cell Signaling Molecules
• Cell-cell recognition
• Although cells can act as self-contained units, they
don’t exist in isolation
• Even a unicellular organism must detect and respond to
outside influences e.g. chemicals, light and other cells
• In a multicellular organism, the organisation of tissues
and systems brings more complexity
• Therefore, it is essential that cells can COMMUNICATECOMMUNICATE
to enable their activities to be fully coordinated

• Communication involves transmitting and receiving
information
• A SIGNALINGSIGNALING cell sends a signal and is received by a
TARGETTARGET cell
[Signal molecules can induce different responses in their target
cells e.g. acetylcholine: causes cardiac muscle to relax, but skeletal
muscle to contract ]
• If a change in the form of a signal is required, it is
called a SIGNAL TRANSDUCTIONSIGNAL TRANSDUCTION
Analogy: Faxing a letter –
conversion of a printed form of
information into an electronic form –
back into a printed form
ANIMATION

Communication Systems
ENDOCRINEENDOCRINE
Secretion of a hormone into the bloodstream for dispersal.
The signalling cell and the target cell can be far apart.
Very slow method e.g. Insulin, Adrenaline
PARACRINEPARACRINE
Secretion of a local mediator. This affects cells in the
immediate area of the signalling cell e.g. Histamine
NEURONALNEURONAL
Nerve cells or neurones elicit responses by the release of
a neurotransmitter at synapses. Can signal over very long
distances via a network of nerve cells. Very fast signalling
e.g. GABA (Gamma-Amino-Butyric-Acid – an inhibitory
neurotransmitter)
CONTACTCONTACT
DEPENDENTDEPENDENT
Signal molecules in the plasma membrane of the signal cell
interact with membrane bound receptors on the target
cell. These signals are therefore restricted to cells which
are in direct contact

Extracellular HYDROPHOBIC Signaling
Molecules
• Some small hydrophobic molecules can cross the plasma
membrane and enter the cell by diffusion
• Best known classes are the STEROIDSTEROID hormones e.g.
cortisol & testosterone and the THYROIDTHYROID hormones e.g.
thyroxine
• The hormones can diffuse across the plasma membrane
and bind to receptor proteins that are located either in
the cytosol or in the nucleus itself
• They work by activating GENE REGULATORY PROTEINSGENE REGULATORY PROTEINS in the
cell, which stimulate transcription of particular sets of
genes in the nucleus

The mode of action of cortisol:
Cortisol is a steroid hormone that is released in the body in
response to physical or psychological stress.
The secretion of cortisol induces energy-directing
processes for the purpose of providing the brain with
sufficient energy sources that prepare an individual to deal
with stressors.
In addition to its role as a so-called "stress hormone",
cortisol plays many key roles in almost every physiologic
system.
Regulation of blood pressure, cardiovascular function,
carbohydrate metabolism, and immune function are among
the best known functions of cortisol.
Action of Cortisol animation [Diagram]

Extracellular HYDROPHILIC Signaling
Molecules
• In contrast to the hydrophobic signals, the majority of
signaling molecules are either too LARGELARGE or too
HYDROPHILICHYDROPHILIC to cross the plasma membrane
• The receptor proteins for these signals must therefore
present a binding site to the extracellular environment and
elicit a response in the cytosol
• There are 3 main classes of these cell surface
transmembrane receptors all of which bind extracellular
signal molecules, but generate intracellular responses in
DIFFERENTDIFFERENT ways …

1) ION-CHANNEL LINKED Receptor
• These are also known as chemically-gated ion channels
• They open pores through the protein in response to
binding of a signal molecule
• Ions flow through this ’gate’ generating an electrical
effect
• This type of receptor is found in excitable cells such
as nerve and muscle cells

• A neurotransmitter (e.g. acetylcholine, noradrenaline) binds to this type of
receptor, altering its conformation to open or close a channel (often through
or near the receptor) to the flow of Na2+, K+, Ca2+, or Cl- ions across the
membrane.
• Driven by their electrochemical gradient (i.e. one side of the membrane has
numerous ions, while the other side has few) the ions rush into or out of the
cell, creating a change in the membrane potential due to the positive or
negative nature of the ions.
• This flow of ions through the channel can trigger a nerve impulse, or
alternatively stop one from occurring.

2) ENZYME LINKED Receptor
• Found in all types of cells
• Generate an enzyme activity (usually a KINASEKINASE activity)
on the cytoplasmic end of the protein
• This kinase activity causes the phosphorylation of
other intracellular proteins, thereby activating them

3) G-PROTEIN LINKED Receptor
• Activate a GTP-binding protein (the G-protein) that sets
off a chain of events in the cell
• This group of receptors is the largest known, and many
different signals and responses can be associated with G-
protein activity
• All have the same structural arrangement within the
membrane – known as a seven-pass transmembrane protein
• Several hundred types of receptor are known, which bind
signals as diverse as peptide hormone, amino acids, fatty
acids and neurotransmitters

• On binding the signal, the G-protein is activated by the
binding of GTP
• This activated protein diffuses away from the receptor
protein site and activates its target protein
• This may be an ion-channel protein or an enzyme such as
adenylate cyclase or phospholipase C These
enzymes catalyse the formation of small molecules known
as secondary messengers which trigger the intracellular
response to the original signal transduction event to the
cell surface.
G-Protein animation

The cyclic AMP (cAMP) signal
transduction pathway
• Adenylate cyclase activity generates cyclic AMP
(cAMP), phospholipase C generates Inositol
Triphosphate (IP3)
• Second messengers are important parts of the signal
transduction pathway, and can have many different
effects
• An outline of the cAMP pathway is shown below:
[Insert cAMP pathway diagram]

Signal Transduction
• Very complex area !
• Signals can be of many different types and can act
either by diffusing across the plasma membrane (such
as STEROID HORMONESSTEROID HORMONES e.g. testosterone and NITRICNITRIC
OXIDEOXIDE) or by interacting with a receptor protein on the
cell surface
• The variety of signals, receptors and responses means
that the system of signal reception and transduction
can generate very specific effects in different types
of cell

• The response of a cell to a signal can involve ion flow,
activation of specific proteins, or changes in gene
expression
• These effects can be short-lived, as in the case of the
generation of an action potential, or they may be
permanent alterations that control the developmental fate
of the cell
• It is therefore clear that the idea of a cell as a self-
contained unit is in fact very far from the reality of the
situation - cells are constantly engaged in the exchange of
information in the form of molecular signals and it is this
that enables cells in multicellular systems to function in an
integrated way.

APPLICATIONS OF DNA
TECHNOLOGY
One of the defining features of modern biology is the
extensive use of the technology of gene
manipulation. It is now possible to manipulate DNA
directly to produce recombinant DNA. The
manipulated genes can be replaced back into the
original, or a different, organism to produce
transgenic plants and animals.

Applications of DNA Technology
1) The Human Genome Project: genetic
mapping, DNA sequencing, genome analysis/comparison
2) Human Therapeutics: detecting genetic
disorders, gene therapy
3) Forensic Uses:DNA profiling
4) Agriculture: transgenic plants, production of BST

The Human Genome Project
• The genome of an organism is it’s complete complement of
genetic information
• Completed in 2003, the international Human Genome Project
was a 13-year project coordinated by the U.S. Department of
Energy and the National Institutes of Health.
• Project goals were to …
- identify all the approximately 20,000-25,000 genes in human DNA,
- determine the sequences of the 3 billion chemical base pairs that make
up human DNA
- store this information in databases
- improve tools for data analysis
- address the ethical, legal, and social issues that may arise from the
project.

• The human genome project has been achieved using 3
approaches:
a) GENETIC MAPPING
b) PHYSICAL MAPPING
c) DNA SEQUENCING
• Firstly however, the desired DNA sequences must be
amplified. The process used to do this is the
POLYMERASE CHAIN REACTION (PCR)POLYMERASE CHAIN REACTION (PCR)

The Polymerase Chain Reaction
(PCR)
• Polymerase chain reaction (PCR) is a revolutionary
molecular biology technique for enzymatically
replicating DNA
• The technique allows a small amount of the DNA
molecule to be amplified many times in an exponential
manner
• PCR is commonly used in medical and biological research
labs for a variety of tasks, such as the detection of
hereditary diseases, the identification of genetic
fingerprints, the diagnosis of infectious diseases, the
cloning of genes, and paternity testing.
[Insert diagram]

PCR product compared
with DNA ladder in
agarose gel
DNA ladder (lane 1),
the PCR product in low
concentration (lane 2),
and high concentration
(lane 3).

Stages in PCR
• PCR, as currently practiced, requires several basic components. These
components are:
• DNA template, which contains the region of the DNA fragment to be
amplified
• Two primers, which determine the beginning and end of the region to be
amplified (primers = short lengths of a known DNA sequence)
• DNA-Polymerase, which copies the region to be amplified
• Nucleotides, from which the DNA-Polymerase builds the new DNA
• Buffer, which provides a suitable chemical environment for the DNA-
Polymerase
• The PCR reaction is carried out in a thermal cycler. This is a machine
that heats and cools the reaction tubes within it to the precise
temperature required for each step of the reaction.

The PCR process consists of a series of twenty to thirty
cycles. Each cycle consists of three steps:
• (1) The double-stranded DNA has to be heated to 94-96°C in order to
separate the strands. This step is called denaturing; it breaks apart the
hydrogen bonds that connect the two DNA strands.
• (2) After separating the DNA strands, the temperature is lowered so
the primers can attach themselves to the single DNA strands. This step
is called annealing. The temperature of this stage depends on the
primers and is usually 5°C below their melting temperature (45-60°C)
• (3) Finally, the DNA-Polymerase has to fill in the missing strands. It
starts at the annealed primer and works its way along the DNA strand.
This step is called extension. The extension temperature depends on
the DNA-Polymerase
Taq DNA Polymerase: This is a thermal stable enzyme isolated from
thermophilic bacteria. This enzyme canonly synthesis DNA in one
direction 3’ - 5’

Applications of PCR
1) MOLECULAR BIOLOGICAL RESEARCH - gene screening analysis
(looking for a gene) and DNA cloning (copying particular DNA
sequences)
2) GENETIC MAPPING STUDIES e.g. human genome project,
sequence tagging on genome sites
3) CLINICAL & DIAGNOSTIC USES - screening and diagnosis of
HIV; cancer (detects mutations of oncogenes); genetic disorders
e.g. cystic fibrosis
4) GENETIC IDENTIFICATION and DNA TYPING - forensic and
parentage testing; sex determination of pre-natal cells;
classification of species
5) IDENTIFICATION of TRACE AMOUNTS of DNA - detection of
contamination of foodstuff by: food-borne pathogens, genetically
modified organisms in food products, presence of pork in beef
etc

Nucleic Acid Hybridisation
• Once a gene has been isolated from a complete genome
as a piece of DNA; we may want to know from which
chromosome gene it came from and where that
chromosome is located; or from which cells of the
organism the gene is transcribed; or to test a sample
of human DNA for mutations in the gene suspected of
causing an inherited disease
• All these questions can be answered by taking
advantage of the fundamental property of DNA :
COMPLEMENTARY BASE PAIRINGCOMPLEMENTARY BASE PAIRING

• Remember, the 2 strands of DNA are held together by
HYDROGEN BONDING. These bonds can be broken by
heating to 90o
c or altering the pH
• These treatments release the single strands but DO NOT
break the strong covalent bonds that link the nucleotides
together
• If the process is reversed i.e. slowly lowering the
temperature and bringing the pH back to normal, the
complementary strands will reform double helices - this is
known as HYBRIDISATIONHYBRIDISATION
• Using this technique, particular DNA sequences can be
identified by hybridisation with the aid of a NUCLEIC ACIDNUCLEIC ACID
PROBEPROBE

Nucleic acid hybridization
(A) If the DNA helix is separated into two strands, the strands should
reanneal, given the appropriate ionic conditions and time.
(B) Similarly, if DNA is separated into its two strands, RNA should be
able to bind to the genes that encode it. If present in sufficiently large
amounts compared with the DNA, the RNA will replace one of the DNA
strands in this region

A Nucleic Acid Probe - a short, single-stranded DNA or RNA
molecule that has been radioactively labelled (e.g. 32-phosphate 32
P)
and is used to identify a complimentary nucleic acid sequence

Genetic Linkage Mapping
• Genetic maps are based on the recombination frequency
between genetic markers during MEIOSIS [see Higher notes!]
• These can be used to locate genes on particular chromosomes
and establish the order of the genes and the approximate
distance between them
• This approach relies on having genetic markers that are
detectable
• Genetic markers are any gene that shows variation (different
alleles). These include genes and other DNA sequences such as
microsatellites, which are tandem repeats of units 2-4 bp in
length. These units are also known as short tandem repeats
and are distributed fairly evenly over the genome, and may
even occur within genes.
• Sometimes these are genes that cause disease, traced in a
family by pedigree analysis

• The marker alleles must be HETEROZYGOUS so that
meiotic recombination can be detected
• NB: if 2 genes are on different chromosomes - they are
unlinked and will sort independently during meiosis
• If 2 genes are on the same chromosomes they are
physically linked and a crossover between them during
Prophase I of meiosis can generate non-parental genotypes
• The chance of a crossover occuring increases as linked
genes become further apart. In fact, they may behave as if
they are essentially unlinked
• Genetic mapping is used to produce a picture of the
locations of the marker loci on the chromosome. However,
it doesn’t provide the precise distances between the genes
[Insert Genetic Linkage diagram]

Physical Mapping
• A physical map is a more detailed map of a genetic map
• As with genetic maps, construction of a physical map
requires markers that can be mapped to an exact
location on the DNA
• Physical maps of the genome can be constructed in a
number of ways, all of which aim to generate a map in
which the distances between markers are known with
reasonable accuracy

Restriction Mapping
• Fragments of DNA are made by cutting with restriction
enzymes or endonucleases
• These are enzymes that cleave DNA at certain nucleotide
sequences, thereby generating specific fragments
• The recognition sequences where restriction enzymes are
short (4,5 or 6 base pairs long) sequences that occur at
defined positions in the DNA
• Using a combination of these enzymes and measuring the size
of fragments produced, the ‘puzzle’ can be pieced together to
give the pattern of restriction enzyme recognition sites in
the DNA
• Defined fragments can then be identified either by their size
or using a specific DNA probe to bind to its complementary
map [electrophoresis or nucleic acid hybridisation] diagram

Restriction Mapping : An exampleAn example
The most straightforward method for restriction
mapping is to digest samples of the DNA with a set
of individual enzymes, and with pairs of those
enzymes
The digests are then "run out" on an agarose gel to
determine sizes of the fragments generated. If you
know the fragment sizes, it is usually a fairly easy task
to deduce where each enzyme cuts, which is what
mapping is all about

Restriction Mapping : An exampleAn example
To illustrate these ideas, consider a plasmid that contains a
3000 base pair (bp) fragment of unknown DNA. Within the vector,
immediately flanking the unknown DNA are unique recognition sites
for the enzymes Kpn I and BamH I. As illustrated in the diagram
below, consider first separate digestions with Kpn I and BamH I :

– Digestion with Kpn I yields two fragments: 1000 bp and "big".
Since there is a single Kpn I site in the vector, the presence of
a 1000 bp fragment tells you that there is also a single Kpn I
site in the unknown DNA and that it is 1000 bp from the Kpn I
in the vector. The "big" fragment consist of the vector plus the
remaining 2000 bp of the unknown
– Digestion with BamH I yields 3 fragments: 600, 2200 and
"big". The "big" fragment is again the vector plus a little bit
(200 bp in this case) of unknown DNA. The presence of 600 and
2200 bp fragments indicate that there are two BamH I sites in
the unknown. You can deduce immediately that one BamH I site
is 2800 bp (600 + 2200) from the BamH I in the vector. The
second BamH I site can be in one of two positions: 600 or 2200
bp from the BamH I site in the vector
At this point, there is no way to know which of these alternativeAt this point, there is no way to know which of these alternative
positions is correctpositions is correct

• The trick to determining where the second BamH I site is located is to
digest the plasmid with Kpn I and BamH I together
• This so-called double digest yields fragments of 600, 1000 and 1200 bp
(plus the "big" fragment). The 600 bp fragment is the same as obtained
by digestion with BamH I alone. The 1000 and 1200 bp fragments tell
you that Kpn I cut within the 2200 bp BamH I fragment observed when
the plasmid was cut with BamH I alone
You already know where Kpn I cuts in the unknown DNA, and you
therefore now know the location of the second BamH I site!

Chromosome Walking
• Used to locate genes or other DNA sequences on a physical map
or to locate genes associated with disorders
– The marker DNA and target DNA must be linked
– DNA probes used to locate and isolate multiple copies of DNA
that have complementary sequences of DNA to the probe in
libraries
– 2 libraries are made, one from cloned fragments of the marker
and one from cloned fragments of the target DNA
– Different restriction enzymes are used so that the fragments
in each library are different but overlap

Gel Electrophoresis
• Since nucleic acids are negatively charged,
they migrate toward the positive pole in an
electric field
• When the electric field is applied through
the gel, molecular sieving takes place.
Shorter chains move faster than longer
ones. Thus, the chains are spread out in the
gel according to their size.
• Double-stranded DNA can be visualized by
adding ethidium bromide, a flat aromatic
chemical that fits between base pairs in the
double helix. Only when bound to DNA does
the ethidium bromide fluoresce orange when
irradiated with UV

DNA Sequencing
• The final stage of the genome project is to determine
and assemble the actual DNA sequence itself. For this
to happen:
– DNA fragments must be generated
– The sequencing technology must be accurate and
fast
– Computer hardware/software must be available to
analyse the data

DNA Sequencing cont.
• The technique used for sequencing is the Dideoxy
Chain Termination method as developed by F. Sanger in
the 1970’s
• This method relies on making a copy of the chosen
DNA template
[See Student monograph for a more comprehensive
explanation – pg.154 - 157]

Comparative Genome Analysis
• In addition to mapping the human genome, the genomes
of other species are also being mapped. These include
species important to biological research and
agriculture such as the mouse, chicken, pig, cow, rice,
wheat, Caenorhabditis elegans (nematode), Drosophila
melanogaster (fruit fly), Saccharomyces cerevisiae
(yeast), Escherichia coli, and other prokaryotes.
• The genomes of some of these organisms, such as E.
coli, yeast, the nematode and the fruit fly have now
been completely mapped and sequenced. These maps
can be used to locate homologous genes in the human
genome and to help in determining gene function.

• Comparative genome analysis is being used to find out
more about evolution. The number of differences in an
amino acid sequence can be used to calculate the time
since two species diverged from a common ancestor. If
there are lots of differences between the maps, it can be
deduced that the species diverged longer ago than if
there are only a few differences. This type of
information is used alongside other methods of measuring
the rate of evolution.
• Gene maps can be used to predict gene order. If gene X
is found next to gene Y and Z in one species, the
likelihood is that it will be found next to the same two
genes in another closely related species. Comparative
maps will be used to find candidate genes for phenotypes
mapped in species as diverse as chicken and human.

Human Therapeutics
How is our knowledge of DNA technology being used
today in human therapeutics?
• Congenital abnormalities are genetically based
diseases and there therefore inherited
• 2 types:
– MONOGENIC :
– POLYGENIC :
caused by a single gene defect (Cystic
Fibrosis, Sickle Cell Anaemia, Haemophilia)
caused by defects in several genes
(Heart Disease, Diabetes, Obesity, some cancers)

Detecting genetic disorders
Characteristics of a monogenic disease usually begins with
the presentation of disease symptoms
Step 1 : Trace disease through family using PEDIGREE
ANALYSIS to determine if the faulty gene is dominant,
recessive or X-linked (crucial for genetic counselling)
Step 2 : Genetic maps are used to identify genetic markers, co-
inherited along with the disease. Recombination
frequencies give the distance of the marker to the
diseased gene. The marker is then located on a more
detailed physical map. The gene is then tracked down,
characterised and sequenced, leading to accurate
diagnostic procedures and potential new treatments

Cystic Fibrosis (CF)
Affects 1 in 2000
Gene CFTR gene (Cystic fibrosis
transmembrane conductance regulator)
Protein
coded
Membrane protein (1480 a.a. long)
2 transmembrane domains
Regulatory region
2 ATP binding sites
Mutations Defective ion transport systems
Result Epithelial surfaces are not fully
hydrated causing sticky mucus
accumulation in the lungs
Symptoms Inflammation of lung tissue; bacterial
infection; high salt content of sweat
Gene found 1989
Chromosome 7

Duchenne Muscular Dystrophy (DMD)
Affects 1 in 3500 boys
Gene DMD gene
Protein
coded
DYSTROPHIN (3685 a.a. in length)
Mutations The cytoskeleton is not linked with
the muscle cell membrane in muscle
cells
Result Muscle cells become permeable,
extracellular fluid flows in & cells
burst
Symptoms Progressive failure of muscle growth
& wastage leading to weakness,
paralysis & respiratory difficulties
Gene found 1987
Chromosome X

X linked inheritance of
Duchenne Muscular
Dystrophy
Autosomal Recessive
Inheritance
of Cystic Fibrosis

Mini-project
• Using either CF or DMD as a case study, write a
report about the discovery and treatment of the
disease making sure to :
– explain the genetic mutation involved
– describe the methods/tests used to detect genetic disorders
such as Cystic Fibrosis and Duchenne Muscular Dystrophy;
– explain the importance of genetic counselling;
– explain how gene therapy could be used to treat genetic
disorders;
– include some analysis of results of gene therapy trials;
– discuss the legal, moral and ethical issues for the future
http://www.gig.org.uk & http://www.who.int/genomics/elsi/en/

Cell & Molecular Biology

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