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Macromolecules
PES Biology
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Macromolecules
THE CHEMISTRY OF LIFE
Organic Molecules - Macromolecules
 There are two classifications of molecules – organic and inorganic
 Inorganic molecules construct those objects that belong to the physical nature
of the earth. Organic molecules belong to those organisms of the living world.
 Organic molecules can be characterised by the following:
1. are produced by living organisms
2. most of them contain the elements C H O
3. are usually complex in structure and found in living organisms
 Biologists call complex and large organic molecules macromolecules that are
classified into four major groups. They are:
 Proteins
 Carbohydrates especially Polysaccarides
 Lipids (fats and oils)
 Nucleic Acids
 each of these macromolecules (polymers) are themselves made up smaller organic
building blocks called monomers.
monomer
polymer
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M6 – Polysaccharides and lipids are important macromolecules in cells and
organisms.
M11 – Macromolecules are used as energy reserves.
Carbohydrates - Polysaccharides
 Consist of CHO backbone
 Made up of monomers called
monosaccharides (eg glucose)
 Monomers can join together to form
disaccharides (eg sucrose)
 Polysaccharides are a chain of monomers
generally used for storage of energy.
 Examples include starch (in plants),
glycogen (in animals)
 Polysaccharides are used for
sucrose
structural purposes. For example,
cellulose (fibre) is used for construction of the cell wall in plants; and chitin is
the hard outer covering found in insects.
Lipids
 Consist of CHO
 Made up of three fatty acids join to a
glycerol molecule
 Similar to carbohydrates in structure but
fewer oxygen molecules.
 Fats - solids at room temperature,
generally come from animals
 Oils - liquids at room temperature,
generally come from plants
 Incorporated in the structure of the cell
membrane; insulator; storage
M6.1 – Know that polysaccharides, including cellulose and chitin, and
phospholipids, contribute to the structural components of cells and
organisms.
ENERGY RESERVES IN CELLS
 polysaccharides are responsible for the storage of energy in cells
 in animals the specific molecule is glycogen and in plants it is starch
 glycogen is usually stored in the liver or in muscle cells where energy demands
are the greatest.
 lipids are produced in humans when there is excess polysaccharides and are
more a long term storage molecule.
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Energy storage: lipids vs carbohydrates
 Energy stored in lipids is twice as much as that stored in the same weight of
carbohydrates.
 When food is stored as lipids, it does not interfere in the water balance, so
fewer problems with osmosis
 Polysaccharides are easier to convert to simple sugars eg glucose. This is the
main source of intermediate energy in cells
 In plants starch is converted to glucose
 In animals glycogen is converted to glucose.
M6.2 - Know that polysaccharides and lipids contribute to energy reserves in
cells.
M11.1 – Know that glycogen, starch and some lipids are important stores of energy.
THE STRUCTURE OF DNA
M1 – The chemical unit of information in most organisms is DNA
1.
DNA
 DNA is a polymer molecule that is essential to all living organisms.
 It was first found in the nucleus of cells and had acidic properties and hence it
was given the name Deoxyribo Nucleic Acid
 Its monomer is the nucleotide
consisting of a sugar (deoxyribo),
a base (nucleic base) and a
phosphate molecule arranged as
shown below.
 the bases are joined together by
weak bonds called hydrogen bonds
 In DNA there are
four different bases
Adenine (A),
Cytosine (C),
Guanine (G) and
Thymine (T).
 the bases can only
join together with an
appropriate partner.
That is Adenine
only bonds with
Thymine and Cytosine with Guanine which are
called base pairs
 the final structure is known as the double helix as
shown below.
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M1.1 - Model the structure of DNA as a double helix
made up of a sequence of complementary bases joined by
weak bonds. These bases are attached to a sugar phosphate
backbone.
M2 – The structural unit of information in the cell is the chromosome
Chromosomes
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the nucleus contains the genetic information called Deoxyribo Nucleic Acid
DNA is organised into structures called chromosomes
chromosomes are long thin strands not visible until the cell is ready to divide
chromatin can be seen with the stain aceto-orcein under a light microscope
when stained the human chromosomes can be organised into pairs ranging from
pair 1 through to pair 23 = 46 chromosomes. This is called a karyotype - there a 2
human karyotypes
A human male karyotype
 male = 22 pairs of autosomes & X Y (sex chromosomes)
 female = 22 pairs & X X
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Genes
 the chromosome is divided up into lengths or units called –genes
 genes are the units of inheritance which are passed from parent to offspring
 the gene consists of a series of nucleotides joined at their bases which is a code
for a particular characteristic. The number of nucleotides and the order of their
base pairings determine the code. i.e. A T G C
 the specific position of a gene on a chromosome is called its locus
 for every characteristic there is a specific gene. Therefore there are several tens of
thousands of genes on each chromosome.
M2.1 – Know that a chromosome is made up of many genes
Genes and chromosomes
- humans have approx. 30,000 –50,000 genes – called the human genome
- in 1990 the genome project set out to map where each gene was located on the 46
chromosomes by recording the 3 billion base sequences found in the genome. At
the Adelaide Women’s and Children’s Hospital scientists are researching
Chromosomes 16.
- each chromosome has specific genes on it which are only located on that
chromosome.
M2.2 –
Explain that each chromosome has genes specific for that chromosome
making it identifiable
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DNA REPLICATION
M7 - Specific base pairing is the mechanism of DNA replication
M12 – DNA carries genetic information from one generation to the next
The Role of DNA
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DNA contains the code which determines what proteins are made in the cell
Proteins can be used for either structural or functional purposes in the cell
Enzymes are made of proteins which determine the chemical processes in the cell
Therefore a gene codes for a specific enzyme and a specific characteristic
In bacteria there are few genes found on a circular chromosome called the plasmid
In summary
 DNA is organised into groups of base pairs called genes
 Genes code for a protein
 Proteins can be used for structure or enzymes
 Enzymes are the catalysts which ensure that chemical reactions occur to
maintain the function of the cell
Mitosis
 It is necessary during the cell life cycle that the genetic code is accurately
duplicated.
 This will ensure that both daughter cells will receive an exact copy of the genetic
code from the parent cell.
 This is achieved through the process of DNA replication.
DNA Replication
 occurs in the following steps:
1.
The double strands of DNA are unzipped by an enzyme at the bases by breaking
hydrogen bonds between the bases.
A
T
C
G
T
A
C
G
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Free nucleotides with the correct corresponding base join up with the unpaired
bases.
A
T
C
G
DNA template
T
C
A
G
T
A
C
G
New strand of DNA
3.
Eventually the whole chromosome is unzipped and free nucleotides join with the
exposed bases.
4.
The result is two identical strands of DNA, joined together at the centromere, that
appears as a double stranded chromosome. These identical strands are called
chromatids.
This process is a semi-conservative mechanism, as each strand of DNA acts as a
template for a new strand.
M7.1 - Illustrate the mechanism of semi-conservative replication through
complementary base pairing
M12.1 – Understand that DNA is perpetuated by semi-conservative replication.
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PROTEINS
Proteins are important macromolecules in living organisms. They are made up of smaller
units bonded together. These smaller units are called amino acids and the bonds between
them are called peptide bonds. Proteins have two main purposes in a cell:
1. for cell structure
2. as enzymes
Protein Structure
 Primary structure - the amino
acids are bonded together to form a
long polypeptide chain.
 Secondary structure The chain
then begins to coil due to chemical
attractions between the amino acids
 Tertiary Structure – the coil then
bends and folds due to chemical
attractions between the amino acids
 Quaternary Structure – another
polypeptide in the tertiary form
may join to form a larger protein.
N.B. The amino acid sequence in the primary structure determines the three dimensional
structure of the overall protein. The sequence of amino acids is in fact determined
by the sequences on the DNA molecule.
The DNA molecule governs the amino acid sequence via a complicated process
called protein synthesis.
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THE GENE AND PROTEIN SYNTHESIS
M3 – The functional unit of the chromosome is the gene
The information that is necessary to control the cell is found in the structure of DNA
called the genetic code. This is the sequence of base pairs that are arranged to code for a
particular protein or RNA molecule. This section of the DNA is called the gene. The
gene then, is the functional unit of the chromosome.
RNA
 RNA is the other type of nucleic acid,
 RNA contains the base Uracil instead of Thymine
 RNA contains a different type of sugar in the sugar-phosphate backbone (ribose
instead of deoxyribose)
 RNA is a single stranded molecule (it does not form a double helix).
 RNA is shorter than DNA.
 There are 3 types: messenger-RNA (mRNA), transfer RNA (tRNA) & Ribosomal
RNA (rRNA)
M3.1 – Know that a gene consists of a unique sequence of bases that codes
for a polypeptide or an RNA molecules
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M4 – The flow of information from DNA to protein is unidirectional in
most organisms i.e. from DNA  RNA  Protein
Imagine the DNA to be something like an instruction manual for the building of all of
the different proteins the cell will need to operate and survive. Remember the DNA is a
long molecule and lies in a organised jumble inside the nucleus of a cell. Synthesis
(construction) of proteins occurs outside the nucleus in the cytoplasm. How does it
overcome this problem?
If you wanted to bake something in you kitchen and you knew that the recipe you needed
was in a book at the local library, how would you go about getting that recipe? One
logical way would be to go to the library, find the recipe book, photocopy it and bring it
home to the kitchen to use it. You wouldn’t bring the library home, nor would you bring
a photocopy of the whole library home – just one copy.
The cell uses similar logic to overcome its problem. A messenger (usually a hormone)
goes to the nucleus and attaches itself to a segment of DNA. This usually starts (initiates)
the copying of the DNA to mRNA. The mRNA then moves out of the nucleus and to a
ribosome where the polypeptide is formed.
This process of synthesising proteins occurs in two stages:
Stage 1 – Transcription – in the nucleus a copy of a segment of DNA is made
Stage 2 – Translation – in the cytoplasm, the copy is then used to construct a
protein
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Stage 1 - Transcription
This is the stage at which a section DNA is copied from the gene so that the message can be sent to the
ribosome. RNA, which is a similar molecule to DNA, is used to copy the DNA.
It occurs in the following way:
1. Protein synthesis starts with the DNA in the nucleus.
2. A protein molecule called RNA Polymerase ‘unzips’ (untwists) the appropriate segment of the DNA
double helix for the transcription process to occur.
3. As the hydrogen bonds are broken between the two strands, the nucleotide bases are exposed. Other,
free nucleotides that are present in the nucleus line up adjacent to the template strand and form a
new strand - the messenger RNA (mRNA for short).
4. As the DNA is copied into the form of a mRNA molecules, the thymine nucleotide is substituted
for a URACIL nucleotide.
5. The RNA polymerase is attached to this new strand and drags it along as it unzips and then copies
the original DNA.
6. Behind the RNA polymerase the unzipped DNA rejoins and retwists back into the double helix
structure again.
7. When the segment has been copied, the mRNA moves out into the cytoplasm through a pore in the
nuclear membrane.
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Stage 2 - Translation
The code that has been copied must now be translated so that the correct sequence of amino acids can
be put together so that desired protein can be produced.
1. mRNA enters the ribosome which is made of rRNA
2. A new type of RNA (transfer RNA or tRNA) enters the ribosome carrying an amino acid.
3. The tRNA moves in and its anti-codon attaches itself to a complementary sequence codon on the
mRNA.
For example, if the base sequence on mRNA is AUG, the complementary sequence on the tRNA is
UAC.
4. A second tRNA moves into the ribosome and attaches itself to the next codon on the mRNA strand.
5. Whilst still attached, enzymes join the two amino acids together.
6. The first tRNA is now free to leave the ribosome and find a similar amino acid in the cytoplasm.
7. Further tRNA molecules move into the ribosome, attach themselves to the mRNA strand, and their
amino acids are joined to the first two.
8. Eventually a stop
sequence (UAA or
UAG) on the mRNA
is reached and no
more amino acids
are added.
9. A new polypeptide
chain has been
formed. It will
undergo twisting
and folding until the
secondary and
tertiary structures
are formed. If other
proteins join it, a
quaternary structure
is formed.
Things to note:
 The instructions on the mRNA are read in sequences of three bases (called base triplets or a codon)
 The tRNA contains a sequence of three bases that complements the codon – this is called the anticodon.
 Each sequence of the codon on the mRNA is used to determine what amino acid the complementary
tRNA carries.
For example:
If the sequence on the mRNA is CCU then the complementary tRNA will carry the amino acid
called "proline".
If the sequence on the mRNA is GUA then the complementary tRNA will carry the amino acid
called "valine".
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The full list of which mRNA codons code for which amino acid is on page 7 of your book.
M4.1 – Describe and illustrate the processes of transcription and
translation, including the roles of mRNA, tRNA, and ribosomes.
M3.2 – Describe how three bases, called a codon in mRNA, code for one
amino acid
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THE 3-D SHAPE OF PROTEINS
M5. - The three dimensional structure of a protein is critical to its function
Proteins:
 consist of CHONS
 are made of a series of amino acids joined together to form a polypeptide
chain
 There are about 20 different amino acids. Therefore this variety and
different lengths of chains will produce a large number of different proteins
 Functions include:
 structural
hair, nails and ligaments
 enzymes
catalysts for reactions
 contraction fibres in muscles
 transport
haemoglobin
 defence
antibodies
 coordination hormones
 storage
albumin in eggs
 Remember - it is the sequence of amino acids that will determine the way a
polypeptide chain folds. It is this folding which give a protein its specific
three-dimensional shape that in turn gives it specific function that may
include binding with other molecules.
 For proteins to carry out their function they must fit with another molecule
perfectly – like 2 pieces of a jigsaw puzzle.
M5.1 – Explain how the three-dimensional structure of proteins can facilitate
recognition and binding of specific molecules, including enzymes and
substrates, and cell membrane receptors and hormones
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ENZYMES
M8 - Enzymes are specific for their substrate
Enzymes
 are biological catalysts – speed up chemical reactions without becoming involved in
the reaction itself.
 become involved in a typical chemical reaction in the following way:
 made up of proteins which have a specific active site
 have an active site is part of the unique 3-D which an enzyme attaches itself to
substrates (reactants).
 are specific – one type of enzyme for one substrate due to structure of active site.
 there are 1000’s of different reactions that take place in a cell therefore there are an
equivalent number of enzymes.
 enzymes are named by adding “ase” to the end of the substrate that it reacts with. E.g.
carbohydrase, protease, lipase, sucrase
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“Induced-fit” model
 when the substrate binds to the enzyme’s active
site, weak hydrogen bonds form the enzyme
substrate complex
 the enzyme changes shape slightly so that the
active site fits exactly into the substrate
 Substrate bonds are stressed by the enzymes
shape and cause the bonds to break more easily,
catalysing the reaction. This is called the
“induced-fit” model
 the enzyme then breaks free and can be re-used
 two major groups of enzymes:
a) intracellular – inside cells
b) extracellular –outside of cells
M8.1 – Describe the induced-fit model of
enzyme substrate binding
FACTORS AFFECTING ACTION OF ENZYMES
Temperature
 Enzymes operate in a strict temperature range
 In humans it is between 35 – 40oC which is the optimum temperature for the rate of
reaction of enzymes
 When temperatures exceed 50 oC enzymes begin to denature – causes the protein to
distort and prevent the active site from joining with substrate
 Low temperatures do not allow for collisions amongst molecules to occur
Enzyme
activity
10
20
30
40
50
Temperature oC
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Chemical Inhibitors
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Inhibitors are chemicals that inhibit the
action of specific enzymes
Some inhibitors compete with substrates
for the active site of the enzyme others
will bind to other parts and distort the
shape of the enzyme
E.g. cyanide, arsenic, DDT
pH
 Enzymes optimum pH varies –
most human enzymes prefer pH
between 6 – 8 but there are
exceptions. Pepsin in the
stomach likes a pH of 2
 However the pH can not vary
greatly otherwise the enzyme
will cease function
Co-factors
 Many enzymes require non-protein co-factors to assist their action
 They bind to the active site of with substrate to allow the enzyme to function
normally
 Some co-factors are organic and are called co-enzymes
 E.g. co-factors - copper, iron, zinc coenzymes - vitamins
M8.2 – Explain how pH, temperature, and chemical inhibitors can alter the
binding of enzymes and substrates at the active site.
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MOLECULAR RECOGNITION
M9 – Molecular recognition is an important property for life processes
 One of the most fundamental aspects of life is the capacity of a cell to be selective in
which chemicals are exchanged with the environment – achieved by the cell
membrane
 Proteins found embedded in the bi-lipid layer allow for this selectivity of chemicals
outside of the membrane. These proteins are sensitive to chemicals, which are
required by the cell and those, which are not.
Example – The action of hormones - Adrenalin
 Hormones are chemicals produced by
“endocrine glands” involved in communication
Hormones
systems in organisms.
 A hormone has a shape that complements one of
the types of receptor proteins found on the
surface of the membrane.
 When the hormone binds to the protein it may
cause a change in the configuration of the
receptor protein, which sets in motion changes in
the cytoplasm.
 An example of this process can be explained with the affect of the hormone adrenalin
– adrenalin when released by the adrenal gland binds to protein receptors on
liver cells.
– This in turn activates series of steps to eventually convert glycogen to
glucose.
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Self And Non-Self Concept
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Our own cells are able to identify which cells are its own (“self”) and which are
foreign (non-self) by the use of their receptor molecules.
Cells which are “self” are identifiable because each of us has our own unique protein
called an antigen lining the surfaces of our cells. Therefore all other organisms,
except for an identical twin, have different antigens.
Being a protein an antigen has its own unique 3-D shape.
White blood cells patrol our body for cells “lacking” this specifically shaped protein
on the outer membrane. When they find these foreign cells (e.g. bacteria) they engulf
and digest them which is the beginning of an immune response.
M9.1 – Explain how cell membrane receptors allow cells to recognise and select
molecules necessary for cell activities.
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ENZYMES AND ACTIVATION ENERGY
M10 – Enzymes increase reaction rates by lowering the activation
energy.
 Chemical reactions involve the breaking and reforming of chemical bonds.
 Reactant molecules must absorb energy for their bonds to break.
 This initial energy required to break bonds to start a reaction is called the activation
energy. (see diagram below)
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 There are two main types of reactions:
-
Endergonic – where the final product
has more energy than
the reactants
(synthesis)
-
Exergonic – the products have less
energy than the
reactants (breakdown)
A reaction requires an initial amount of energy but once it is started then the
reaction proceeds quite rapidly.
M10.1 - Understand that reactions require an initial input of energy to proceed.
 Biological enzymes have evolved to speed up the rate of reaction by lowering the
activation energy (see diagram above)
 This can be achieved by:
a) During the induced fit phase of enzyme action, the enzyme puts the chemical
bonds under pressure, making them easier to break.
b) Enzymes may hold substrates in the correct orientation so that they can react
quicker.
 Enzymes are not consumed in the reaction and so can be re-used.
 They may typically convert something in the order of thousands of substrate
molecules per second.
M10.2 – Know that enzymes catalyse biological reactions by lowering the input of
energy required.
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MUTATIONS
M15 - Change in the base sequence of DNA can lead to the alteration or absence of
proteins and to the appearance of new characteristics in the descendents.
 We have seen that the sequence of bases on the DNA molecule directs the assembly of
proteins.
 We have also seen that these proteins due to their unique 3-D shape have special
functions.
 Changes in the sequence of bases in the DNA are called mutations.
 A change in the sequence of DNA bases can affect the sequence of amino acids and
therefore the 3-D structure of the protein.
 The effects of mutations can range from negligible to fatal.
 Mutations are random, frequent and increased by various factors.
 Mutations are permanent changes to DNA and are ultimately the source of any new
genes.
These are the following categories that can alter the nucleotide sequence:
Base-Pair Substitutions
- where one nucleotide is
substituted for another –
wrong base is placed into
position on the DNA
Base-Pair Insertions or Deletions
- the addition or deletion of
nucleotides on DNA
- can have disastrous
impact when translation
occurs
- outcome could be the
production of a new
polypeptide sequence
M15 .1 – Know that changes in the DNA sequence are called mutations
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Mutation Rates
Factors that increase mutations are:
1) Radiation (UV, x-ray etc)
2) Mutagenic chemicals (benzene, tar)
3) Heat
All can lead to changes in the genes that control cell division and ultimately lead
to cancerous cells.
M15.2 – Know that the mutation rate can be increased by radiation, muatgenic
chemicals and heat
Effects of Inheritable mutations
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
mutations are changes that occur in DNA which can be inherited by the next
generation if they have taken place in the gametes of the parent
most mutations are harmful and can cause diseases such as cystic fibrosis and sicklecell anaemia to be inherited. These two diseases are a result of a single mutation at
one nucleotide.
Sickle-cell anaemia
 This mutation causes the haemoglobin (protein) in blood to from abnormally and
gives rise to a blood cell which is sickle shape
 There fore people with this condition are anaemic and their blood often clots because
of the shape of the blood cells.
 Haemoglobin is made up 2 pairs of polypeptide chains; one contains 141 amino acids
and the other 146.
 In people suffering from sickle-cell anaemia, in one of the chains glutamic acid is
substituted with valine
 This one change in amino acid sequence leads to the disease
DNA Template
DNA Complementary Strand
MRNA Molecule
Amino Acid Sequence
Type of Haemoglobin
Type of Red Blood Cells
Effect on Behaviour
Normal Individual
-TCTGGGCTCCTTTTT-AGACCCGAGGAAAAA-UCUGGGCUCCUUUUUThreonine-Proline-Glutamic AcidGlutamic Acid-Lysine
Normal
Normal
Alive
Sickle Cell Anaemia Sufferer
-TCTGGGCACCTTTTT-AGACCCGTGGAAAAA-UCUGGGCACCUUUUUThreonine-Proline-Valine-Glutamic AcidLysine
Abnormal
Sickle Cell
Short Life Span/Death
M15.3 – Explain how inheritable mutations can lead to changes in the
characteristics of the descendents.
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THE ROLE OF DNA IN EVOLUTION
M13 - The universal presence of DNA is strong evidence for the common
ancestry of all living things
DNA and living organisms
 Nucleic acids are the only known molecules that are able to both store and transmit
information so that the cell can carry out its functions and reproduce.
 All living cells contain nucleic acids, which is strong evidence that all life as we know
it has the same origin.
 The diversity of life today has taken billions of years, which can be explained by
mutations.
 Mutations alter the base sequences of DNA. The altered gene can then be passed on to
offspring to produce characteristics, which are favourable or fatal.
 These mutations are then passed on from generation to generation.
M13.1 – Know that DNA is universal to most living organisms
EVOLUTION
Definition – “A theory regarding the progressive changes in species over a long period of
time.”
i.e. that species have evolved from simpler forms that lived in the past.
 We have discussed the universal presence of DNA as being strong evidence for the
common ancestry of all living things. DNA is the fundamental chemical of all living
things.
 The enormous number of possible combinations of nucleotide sequences allows for
the variety of structure (biodiversity) observed in living things today.
 The theory of evolution explains the transformation of life from the simplest of
bacteria to the diversity of today. The theory of evolution has developed from the
ideas of Charles Darwin, which he published in his book "The Origin of Species”
 An organism's characteristics are determined by the 3-D structure and function of its
proteins, which are ultimately coded for by the DNA.
 The DNA sequence is modified by mutations which are rare events and even when
they do occur they are rarely advantageous and will kill the organism before they
have the chance to pass it onto their offspring. It is therefore understandable that the
extraordinary biodiversity observed today developed over billions of years.
 DNA is responsible for the features and inheritable characteristics of all living things.
 Darwin compared anatomical features of different species and noted that whilst they
had different functions (e.g. flying, grasping and walking) they were similar in
structure.
 These structures suggest that there is some common origin. That is they have similar
DNA sequences.
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In summary
The study of DNA structure in recent times has lent much support for the theory of
evolution.
 The fact that all living organisms contain DNA strongly suggests that all life forms
have a common ancestor. (The first type of organism being a prokaryote around 3.4
billion years ago)
 If not we would expect different life forms to have different systems
 The sequence of DNA has been gradually modified and diversified over billions of
years.
 Evolution is characterized by the changes in structures of living organisms over time.
 The only way that characteristics could have changed is through changes in DNA
structure (mutations).
M13.2 – Know that DNA has diversified over billions of years
THE EVIDENCE FROM DNA
M14 – DNA and protein sequences usually show greater similarity
between closely related groups of organisms than between
distantly related organisms.
Question –
Primates are said to have a common ancestry (related) to humans. If you were
a geneticist what evidence would you look for to prove that there was some
common element between humans and primates?
Answer –
 If there is a similarity in appearance there must be some similarity in proteins
(enzymes) between the two species
 If proteins are similar there must be similar types of genes therefore similar types of
DNA sequences.
 Therefore to have these similarities one explanation is that humans and primates
somewhere in time, shared a common ancestor.
 When comparing the features of different organisms, we can now look beyond their
major organs and limb structure. Scientists now have the technology to distinguish
similarities in the DNA of different species.
 Similarities between different organisms are linked to similarities in their DNA and
protein molecules.
 We now have the technology to sequence the amino acids in protein molecules. If the
sequence of amino acids from the proteins of two different species is found to be
similar, then it follows that the sequence of bases in their DNA must also be similar.
 If the nucleotide sequence is simi1ar then it is logical to infer that they inherited it
from a common ancestor.
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Macromolecules
PES Biology
Cytochrome C - An example to explain
 Respiration is the process of harvesting the chemical energy of food molecules.
Respiration is therefore a vital process for life.
 A protein called cytochrome c is involved in the respiration reactions of virtually all
living things and is therefore a useful protein to sequence and use to compare the
DNA of different organisms.
 This protein varies from one species to another – the degree of similarity indicating
how closely related the two organisms are. The comparisons of amino acid sequences
from cytochrome c of a variety of organisms is illustrated in a phylogenetic tree
 When the DNA of two different species is compared and determined to have similar
sequences, the two species are said to have a recent common ancestor.
 The greater the amount of time the two species have been separated, the more chance
there has been for mutations to occur. This means that two species with large
differences in their DNA are in fact distantly related.
M14.1 – Understand that organisms have common features attributed to commonly
shared sequences of DNA
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Macromolecules
PES Biology
DNA HYBRIDIZATION
How can we test to see how much of a difference there is between DNA sequences?
 DNA hybridisation is a method used to compare the DNA of different species.
 The process involves heating the DNA of two different species so that the weak
hydrogen bonds break between the two complimentary strands and they separate
from one another.
 The strands of two species are then mixed and allowed to cool in the presence of one
another.
 During the cooling process, the DNA recombines and attempts to form the double
helix once again.
 The more regions containing complimentary sequences between the. DNA of the two
species there are, the stronger the two strands are held together.
 The newly formed DNA is now reheated to see how readily the two strands separate.
 DNA from closely related' species will have more regions which are similar and will
therefore be held together with greater strength and separate at a higher temperature
than DNA that is poorly matched. (The separating temperatures are compared).
Conclusions
 Good matches indicate much of the DNA in
common with each other and thus the closer
evolutionary relationship between species
 This closeness suggests that the two species only
recently separated from each other.
M14.2 – Explain why the greater the similarity between the sequence of nucleotides
in their DNA, the more likely it is that the separation of two species is
recent.
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Macromolecules
PES Biology
MANIPULATION OF DNA
M16 - Human beings can manipulate DNA
 Once the role of the sequence of nucleotides in DNA as genetic information was
established, it was' only a matter of time before the technology to manipulate this
information was developed.
 Genetic engineering or genetic modification is possible because we have the
technology to isolate and manipulate genes that have been extracted from the DNA of
species.
 Essentially, genes are taken from one organism altered, and returned to the original
organism or remain unaltered but are placed in another organism. In some cases, a
specific segment of DNA is artificially synthesised and transferred to another
organism
 The implications of this technology are mind-boggling.
 Simple organisms such as bacteria and yeast are often used in genetic engineering
because of the nature of their mode of reproduction. They multiply (thus replicating
their DNA) rapidly
 Scientists can transfer genes into bacteria and yeasts, which then simply and rapidly
clone the gene along with the rest of their original DNA and then produce the protein
coded for by the gene.
 Because DNA from different sources are being combined, the process is (more
accurately) recombinant DNA technology
DNA CAN BE EXTRACTED FROM CELLS
Scientists can extract DNA from cells by breaking the cells, separating the nuclei (via
centrifuging techniques), removing the nuclear membrane with special chemicals and
then isolating the DNA from the nuclear proteins.
IDENTIFYING AND ISOLATING THE DESIRED GENE
 Once the DNA has been extracted, restriction
enzymes are then used to the cut the long DNA
strands into smaller fragments.
 Restriction enzymes can cut DNA at specific
sites. The sequence, of bases recognised by a
restriction enzyme is called a restriction site
and usually consists of 4 – 6 nucleotides.
 Some restriction enzymes cut directly, across the
DNA strand, leaving 'blunt' ends, whilst others
cut diagonally across the DNA strand, leaving
some of the bases exposed (called 'sticky'
ends). The fragments are still double stranded.
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Macromolecules
PES Biology
 There are 1000's of known restriction enzymes all of which have been isolated from
bacteria. Bacteria have evolved these enzymes and are protected against viruses
(which insert harmful DNA).
 Now that the DNA has been extracted and cut into smaller segments, the desired gene
must be isolated' from the fragments of DNA. There are two ways to achieve this:
1. ANTIBODIES
2. PROBES
ANTIBODY METHOD
 The protein that is coded for by the desired gene is injected into an animal.
 The animal then responds by making antibodies that bind to the protein in a specific
and complimentary manner. These antibodies are then collected and labelled
(radioactively or with chemicals).
 The DNA from the cell (now in fragments from the restriction enzymes) is then
incorporated into huge number of bacterial cells. One bacterial cell will incorporate
only on or two fragments.
 The principle behind recombinant DNA technology is simplified in the diagram
below.
(A plasmid is a small ring of DNA
that carries accessory genes
separate from those of a bacterial
chromosome)
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Macromolecules
PES Biology
 So the situation now is that we have huge amounts of bacteria, each containing
different genes derived from the DNA fragments.
 These bacteria are then incubated at their optimum temperature and allowed to
multiply, as they multiply, colonies of each of the original bacteria are formed.
 These colonies use the incorporated DNA fragments (one of them contains the
desired DNA fragment/gene) to synthesise the desired protein.
 The labelled antibodies are then added to each of the colonies
 Only the colony with the desired gene will be able to produce the protein that will
bind in a specific and complimentary manner to the labelled antibodies (like a lock
and key. Scientists can then isolate this colony and clone the gene.
PROBES
 A short segment of RNA or single stranded DNA containing a
sequence of bases that is complimentary to a part of the
desired gene (also a sequence of bases) is selected for this
process.
 This complimentary segment is then radioactively labelled
(now called a probe)
 The DNA that has been extracted from the cells and then cut
into fragments with restriction enzymes is double stranded. It
is now necessary to separate the strands. This is achieved
by heating them.
 The probe molecules are then added to the heated solution
containing separated DNA strands and allowed to cool in
the presence of one another.
 On cooling, some of the probe molecules will bind with
their complimentary segment (located on the desired gene).
Because the probe is radioactively labelled, scientists can
identify and isolate the gene.
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Macromolecules
PES Biology
CLONING DNA
 The DNA from the original cell has been extracted, fragmented (using restriction
enzymes) and the desired gene isolated {either by DNA or RNA probes using the
antibody method).
 Now that the desired gene has been isolated, it is necessary to make numerous copies
and synthesise the protein encoded in the gene.
 A bacterial plasmid is extracted and cut at a specific site with a restriction enzyme,
leaving stick ends.
 The desired gene (with complementary stick ends) is then mixed with these cut
plasmids so that as they recombine (this requires an enzyme called DNA ligase), they
incorporate the desired gene.
 The altered plasmid is then reinserted into a bacterial cell so that as the bacteria
multiply the recombinant gene will be copied and therefore be present in all of the
descendent bacteria. Now, an entire colony of identical bacterial cells are capable of
synthesising the desired protein.
 Such desired proteins may, for example be insulin (for diabetics, vaccines for
diseases (eg hepatitis), growth hormones, enzymes used in laundry detergents and a
variety of useful drugs.
M16.1 – Know DNA can be
extracted from cells
M16.2 – Describe how particular
genes can be selected and removed,
using probes and restriction
enzymes
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Macromolecules
PES Biology
TRANSGENESIS
 It is also possible to incorporate DNA into cells other than
bacteria. Animals and plants grown from genetically modified
DNA are called transgenic and the technique is known as
transgenesis
 Numerous quantities of the desired gene are injected into a
fertilised ovum (egg) by microinjection. This process does not
have 'a 100% success rate. (Thus the reason for numerous
quantities of the gene being injected), but
it is hoped that the cell's DNA will
incorporate a copy of the gene. This way,
when the embryo is returned to the mother
and allowed to develop, each cell in the
resulting adult organism will contain the
gene. (see summary diagram right)
 Useful proteins can be made by goats and secreted in their milk
(e.g. a protein used by doctors to dissolve blood clots in coronary
arteries thus reducing heart attacks) or transgenic cotton plants
now contain a gene which codes for their very own pesticide (It
would no longer be necessary to use crop dusters and pollute
river systems!)
 Many genetic diseases may be able to be treated using gene
therapy. By inserting a normal gene into cells that can then be
implanted into the patient, a tissue that functions normally could
be formed.
 With DNA technology we can now produce a type of protein
(antigen), which normally occurs on the surface of harmful
invading pathogens. This would not impose any danger as only
the protein, (and not the accompanying cell) would be injected
into the bloodstream. This would stimulate an immune response
and the body could then build up an immunity (produce
antibodies) without ever experiencing any of the
harmful symptoms.
M16.3 – Explain how some selected genes can be
transferred between species.
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Macromolecules
PES Biology
SOCIAL CONSEQUENCE OF GENETIC MANIPULATION
Great medical advances have been made and there is more promise to follow as a result
of recombinant DNA technology. Over the past decade humans have benefited from the
medical applications of genetic engineering, improved food products and improved
world production of crops.
The ability of humans to manipulate DNA does however raise some serious ethical
issues. Whilst some of the applications of genetic engineering are apparently harmless,
there is every chance scientists may unintentionally create a gene that is harmful and in
fact endanger organisms.
One might argue however, that DNA has been altered (mutations) naturally over
billions of years of evolution and that genetic engineering is only accelerating this
process.
GENETIC SCREENING OF ADULTS & EMBRYOS Humans can have their DNA
or even their embryo's (pre-pregnancy) DNA screened for certain diseases and
characteristics. Important decisions can be made based on the information obtained from
an embryo's DNA - "Should/shouldn't we return this embryo to the womb?
SUFFERING OF ANIMALS There is a genuine concern for the suffering of animals
involved in genetic engineering research.
ECOLOGICAL CONCERNS How do we know what will happen in the field once we
take this technology away from the controlled environment of the laboratory? There may
be unknown toxic effects of transgenic products. Some transgenic crops may grow so
successfully that they out compete the native vegetation, destroying the habitat and
reducing biodiversity.
LAWS AND POLICIES Laws and policies spend years on the desks of bureaucrats,
before they are changed. It is difficult to come up with sensible and moral policies
regarding the accepted practice in this new area.
MONOPOLIES HELD BY LARGE COMPANIES. Large companies already hold
patents to processes and products. They are likely to increase their control and profit at
the expense of people wishing to use new techniques.
Consider What if…
 People are able to clone themselves to provide a supply of organs for their future use?
 Billion year old processes are being altered for the purpose of short term gains?
 Profits of industry occur to the detriment of ecosystems and the biosphere?
 People’s genetic code is available to various organizations?
 Variation to disease resistance is lost as individuals within a species become to
similar?
M16.4 – Discuss the social consequences of the manipulation of DNA
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Macromolecules
PES Biology
DNA SEQUENCING
M17 - Human beings can sequence even small amounts of DNA
 Segments of DNA can have their base sequences identified. Fred Sanger was
awarded a Nobel Prize for developing a process to achieve this.
 Samples of DNA are artificially replicated. The nucleotides required for replication
are supplied, as is the enzyme responsible for DNA replication (DNA polymerase).
DNA polymerase unites specific and complementary free nucleotides with the
template strands.
 Modified versions of each of the four nucleotides (A, T, G, & C) are then added to
different test tubes containing the replicating DNA. As the DNA-polymerase binds a
modified nucleotide to the template strand, it is unable to add further nucleotides to
the template strand and replication stops.
 The scientist conducting the process has a record of which type of modified
nucleotide was added to the test tube and stopped the replication.
 The lengths of the DNA strands from each test tube can then be compared and the
site where the replication stopped indicates which bases are present.
THE POLYMERASE CHAIN REACTION (PCR)
 The polymerase chain reaction procedure is used to essentially multiply DNA
samples. The process of DNA replication is carried out artificially in a laboratory and
serves to amplify small samples of DNA (eg. from a crime scene) to a point where
there is enough DNA to analyse by DNA fingerprinting.
 The sample DNA is added to a test tube and heated so as to separate the
complementary strands. These single strands will now serve as template strands in
the next part of the process.
 DNA polymerase is added to unite the specific and complementary free nucleotides
with the single strands so that on cooling, the double helix forms once more. Now the
sample has doubled. In approximately one hour and after about 20 PCR cycles, it is
possible to multiply the sample DNA by a million times.
 Before the invention of PCR, the only way to make multiple copies of DNA in a
short time was to use produce recombinant bacterial plasmids and clone the bacteria.
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Macromolecules
PES Biology
How the PCR technique occurs
M17.1 – Understand that segments of DNA can be multiplied, using the polymerase
chain reaction (PCR) and then have their base sequences identified
(details are not required).
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Macromolecules
PES Biology
DNA FINGERPRINTING
 Every time your body comes into contact with something, the surface will then
contain small amounts of your cells and their associated DNA.
 PCR technology allows for the sequencing of this DNA. Our DNA sequence is as
unique to us as our fingerprints.
 In the region of the centromere, there is a long segment (up to 25% of the total DNA)
that does not code for anything). This segment contains highly repetitive sequences
(called a ‘junk repeat'). Mutations that occur in this region don't affect the organism,
as the region does not code for any proteins. For this reason, the frequency of junk
repeats is extremely variable from one person to the next.
 How often (the frequency) this sequence is repeated is as unique to an individual as a
fingerprint. For example, I might have the sequence A T repeated 70 times along my
'junk repeat' segment whereas someone else may have it, repeated only 58 times.
Another application...
 Samples of DNA can be broken up into fragments using restriction enzymes.
Remembering that restriction enzymes cut-the DNA at specific sites (sequences) and
considering that the DNA sequence for everyone is different, the length of the
resulting fragments will also be different from person to person,
 These different length fragments are called Restriction Fragment Length
Polymorphisms (RFLP'S). The RFLP's are then separated according to their size,
which creates a banding pattern unique to the individual. The odds of two unrelated
people, having the same banding pattern are 100,000,000 to 1. Lawyers and scientists
are working to ensure that the tests are free from misinterpretation.
 DNA fingerprinting can be used for medicinal purposes (to detect viral DNA in the
blood that causes disease or genetic defects in human embryos) and in forensic and
fossil research.
 DNA fingerprinting can be used as evidence in custody disputes for determining
genetic, relationships and is also a more accurate way of identifying criminals than
blood typing or fingerprints. DNA that has been left for 3-4years can in fact still be
used to produce a DNA fingerprint
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Macromolecules
PES Biology
Negative
Electrode
Wells where
three samples of
DNA have been
placed
DNA samples
move towards
Positive electrode
Smaller fragments
move further than
larger fragments
Positive
Electrode
M17.2 – Explain how differences in DNA sequences, identified by DNA
fingerprinting, can be used in forensic science.
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