Module 2 Notes

Module 2 - Genetics - page 1
AQA(B) AS Module 2:
Genes and Genetic Engineering
Contents
Specification 2
DNA Nucleotides 4
DNA Structure 6
DNA Function 7
Replication 8
RNA 10
Transcription 11
The Genetic Code 13
Translation 14
Mutations 16
The Cell Cycle DNA and Chromosomes 19
The Cell Cycle and Mitosis 23
Asexual Reproduction 25
Sexual Reproduction 30
Genetic Engineering Techniques 34
Applications 49
These notes may be used freely by A level biology students

and teachers, and they may be copied and edited. I would be
interested to hear of any comments and corrections.
Neil C Millar (nmillar@ntlworld.com)

Head of Biology, Heckmondwike Grammar School,
High Street, Heckmondwike WF16 0AH
HGS A-level notes NCM/6/07

Module 2 Specification
the same relative position (locus) on homologous
DNA chromosomes.
Structure. DNA is a stable polynucleotide. The
double-helix structure of the DNA molecule in terms Mitosis
of: the components of DNA nucleotides; the sugar- Mitosis increases cell number in growth and tissue
phosphate backbone; specific base pairing and repair and in asexual reproduction. During mitosis
hydrogen bonding between polynucleotide strands DNA replicates in the parent cell, which divides to
(only simple diagrams of DNA structure are needed; produce two new cells, each containing an exact copy
structural formulae are not required). Explain how the of the DNA of the parent cell. Candidates should be
structure of DNA is related to its functions. able to name and explain the stages of mitosis and
recognise each stage from diagrams and photographs.
Replication. The semi-conservative mechanism of
DNA replication, including the role of DNA Asexual Reproduction and Cloning
polymerase. Genetically identical organisms (clones) can be
produced by using vegetative propagation, and by the
Transcription. The structure of RNA. The production splitting of embryos. Given appropriate information,
of mRNA in transcription, and the role of RNA candidates should be able to explain the principles
polymerase. Explain how the structure of RNA is involved in:
related to its functions. • producing crops by vegetative propagation
• the cloning of animals by splitting apart the cells of
Translation. The roles of ribosomes, mRNA and its developing embryos.
codons, and tRNA and its anticodons in translation.
Meiosis
Genetic Code. How DNA acts as a genetic code by During meiosis, cells containing pairs of homologous
controlling the sequence of amino acids in a chromosomes divide to produce gametes containing
polypeptide. Codons for amino acids are triplets of one chromosome from each homologous pair. In
nucleotide bases. Candidates should be able to explain meiosis the number of chromosomes is reduced from
the relationship between genes, proteins and enzymes. the diploid number (2n) to the haploid number (n).
(Details of meiosis not required.)
Mutations
New forms of alleles arise from changes (mutations) in Sexual Reproduction and Gametes
existing alleles. Sexual reproduction involves gamete formation and
• Gene mutation as the result of a change in the fertilisation. In sexual reproduction DNA from one
sequence of bases in DNA, to include addition, generation is passed to the next by gametes. When
deletion and substitution. gametes fuse at fertilisation to form a zygote the
• A change in the sequence of bases in an individual diploid number is restored. This enables a constant
gene may result in a change in the amino acid chromosome number to be maintained from generation
sequence in the polypeptide. to generation.
• The resulting change in polypeptide structure may
alter the way the protein functions. Differences between male and female gametes in terms
• As a result of mutation, enzymes may function less of size, number produced and mobility.
efficiently or not at all, causing a metabolic block to
occur in a metabolic pathway. Sexual Life Cycles
Candidates should be able to interpret life cycles of
Mutations occur naturally at random. High-energy organisms in terms of mitosis, meiosis, fertilisation and
radiation, high-energy particles and some chemicals chromosome number.
are mutagenic agents.
Genetic Engineering
Reproduction In genetic engineering, genes are taken from one
Genes and Chromosomes organism and inserted into another.
Genes are sections of DNA which contain coded • The process of DNA replication can be made to
information that determines the nature and occur artificially and repeatedly in a laboratory
development of organisms. A gene can exist in process called the polymerase chain reaction (PCR).
different forms called alleles, which are positioned, in

• The use of PCR, radioactive labelling and How animals can be genetically engineered to produce
electrophoresis to determine the sequence of substances useful in treating human diseases, as
nucleotides in DNA. exemplified by genetically engineering sheep to
• The use of restriction endonuclease enzymes to produce alpha-1-antitrypsin which is used to treat
extract the relevant section of DNA. emphysema and cystic fibrosis.
• The use of ligase enzyme to join this DNA into the
DNA of another organism. Gene Therapy and Cystic Fibrosis
• Plasmids are often used as vectors to incorporate In gene therapy healthy genes may be cloned and used
selected genes into bacterial cells. to replace defective genes. In cystic fibrosis the
• Genetic markers in plasmids, such as genes which transmembrane regulator protein, CFTR, is defective.
confer antibiotic resistance, and replica plating may A mutant of the gene that produces CFTR results in
be used to detect the bacterial cells that contain CFTR with one missing amino acid. The symptoms of
genetically engineered plasmids. cystic fibrosis related to the malfunctioning of CFTR.
• Rapid reproduction of microorganisms enables a
transferred gene to be cloned, producing many Techniques that might possibly be used to introduce
copies of the gene. healthy CFTR genes into lung epithelial cells include:
• use of a harmless virus into which the CFTR gene
Genetically Modified Microbes has been inserted
Microorganisms are widely used as recipient cells • wrapping the gene in lipid molecules that can pass
during gene transfer. Bacteria containing a transferred through the membranes of lung cells.
gene can be cultured on a large scale in industrial
fermenters. Useful substances produced by using Evaluation of Genetic Engineering
genetically engineered microorganisms include Candidates should be able to evaluate the ethical,
antibiotics, hormones and enzymes. (Details of social and economic issues involved in the use of
manufacturing processes not required.) genetic engineering in medicine and in food
production.
Genetically Modified Animals
Genetics
Genetics is the study of heredity (from the Latin genesis = birth). The big question to be answered
is: why do organisms look almost, but not exactly, like their parents? There are three branches of
modern genetics:
1. Molecular Genetics (or Molecular Biology), which is the study of heredity at the molecular level,
and so is mainly concerned with the molecule DNA. It also includes genetic engineering and
cloning, and is very trendy. This module is mostly about molecular genetics.
2. Classical, Transmission or Mendelian Genetics, which is the study of heredity at the whole
organism level by looking at how characteristics are inherited. This method was pioneered by
Gregor Mendel (1822-1884). It is less fashionable today than molecular genetics, but still has a
lot to tell us. This is covered in Module 4.
3. Population Genetics, which is the study of genetic differences within and between species,
including how species evolve by natural selection. Some of this is also covered in Module 4.

DNA
DNA and its close relative RNA are perhaps the most important molecules in biology. They contain
the instructions that make every single living organism on the planet, and yet it is only in the past
50 years that we have begun to understand them. DNA stands for deoxyribonucleic acid and RNA
for ribonucleic acid, and they are called nucleic acids because they are weak acids, first found in the
nuclei of cells. They are polymers, composed of monomers called nucleotides.
Nucleotides
Nucleotides contain the elements CHONP, and have three parts to them:
phosphate sugar base
-
O
O- P O CHOH
5'
phosphate
O O or more simply:
C sugar base
4'
C1' N
C3' C2'
OH OH
• a phosphate group (PO 42- ), which is negatively charged, and gives nucleic acids their acidic
properties.
• a pentose sugar, which has 5 carbon atoms in it. If carbon 2' has a hydroxyl group attached (as
shown), then the sugar is ribose, found in RNA. If the carbon 2' just has a hydrogen atom
attached instead, then the sugar is deoxyribose, found in DNA.
• a nitrogenous base. There are five different bases (and you don't need to know their structures),
but they all contain the elements carbon, hydrogen, oxygen and nitrogen. Since there are five
bases, there are five different nucleotides:
Base: Adenine (A) Cytosine (C) Guanine (G) Thymine (T) Uracil (U)
Nucleotide: Adenosine Cytidine Guanosine Thymidine Uridine
The bases are usually known by their first letters only, so you don't need to learn the full names.
The base thymine is found in DNA only and the base uracil is found in RNA only, so there are
only four different bases present at a time in one nucleic acid molecule.
The nucleotide above is shown with a single phosphate group, but in fact nucleotides can have one,
two or three phosphate groups. So for instance you can have adenosine monophosphate (AMP),
adenosine diphosphate (ADP) and adenosine triphosphate (ATP). These nucleotides are very

common in cells and have many roles other than just part of DNA. ATP is used as an energy store
(see module 3), while AMP and GTP are used as messenger chemicals (see module 4).
Nucleotide Polymerisation
Nucleotides polymerise by forming phosphodiester sugar-phosphate
backbone
bonds between carbon 3' of the sugar and an oxygen
atom of the phosphate. This is a condensation
polymerisation reaction. The bases do not take part in phosphate
5'
the polymerisation, so there is a sugar-phosphate sugar base
backbone with the bases extending off it. This means 3'
that the nucleotides can join together in any order along

5'
the chain. Two nucleotides form a dinucleotide, three
form a trinucleotide, a few form an oligonucleotide, phosphodiester 3'
bond
and many form a polynucleotide.
5'
A polynucleotide has a free phosphate group at one 3' 2'
OH
end, called the 5' end because the phosphate is attached
to carbon 5' of the sugar, and a free OH group at the new nucleotide
joining chain
other end, called the 3' end because it's on carbon 3' of 5'
the sugar. The terms 3' and 5' are often used to denote
3' 2'
the different ends of a DNA molecule.

Structure of DNA
The three-dimensional structure of DNA was discovered in 1953 by Watson and Crick in
Cambridge, using experimental data of Wilkins and Franklin in London, for which work they won a
Nobel Prize. The main features of the structure are:
• DNA is double-stranded, so there are two polynucleotide stands alongside each other. The
strands are antiparallel, i.e. they run in opposite directions.
• The two strands are wound round each other to form a double helix (not a spiral, despite what
some textbooks say).
• The two strands are joined together by hydrogen bonds between the bases. The bases therefore
form base pairs, which are like rungs of a ladder.
• The base pairs are specific. A only binds to T (and T with A), and C only binds to G (and G with
C). These are called complementary base pairs (or sometimes Watson-Crick base pairs). This
means that whatever the sequence of bases along one strand, the sequence of bases on the other
stand must be complementary to it. (Incidentally, complementary, which means matching, is
different from complimentary, which means being nice.)
5' 3'
C G
hydrogen bonds
A T
G C
T A
C G
C G
3' 5'
DNA showing the DNA showing the spacefilling model
complementary base pairing double helix of the double helix
between antiparallel strands

Function of DNA
DNA is the genetic material, and genes are made of DNA. So what do genes (and DNA) do? There
are two definitions of a gene:
Traditional Definition Modern Definition
A gene is an inherited factor that controls a A gene is a section of DNA that codes for a
particular characteristic (such as flower colour). particular polypeptide.
Surprisingly, these two definitions actually say the same thing, since characteristics are controlled
by genes through the proteins they code for, like this:
shape and
sequences sequence of
determines determines function determines characteristics
of bases amino acids
of protein of cell
in DNA in polypeptide
(e.g. enzyme)
This process of making proteins and so controlling characteristics is called gene expression
(because the gene "expresses" itself). Expression can be split into two parts: transcription (making
RNA) and translation (making proteins). DNA has one other important function: the DNA, with all
its genes, must be copied every time a cell divides by mitosis, so that the daughter cells have
identical copies of all the genes. This copying process is called replication. These functions of DNA
are summarised in this diagram (called the central dogma of genetics).
expression
n
plicatio
transcription translation
RNA Protein
re
No one knows exactly how many genes we humans have to control all our characteristics, but the
current best estimate is around 30 thousand. The sum of all the genes in an organism is called the
genome, and this table shows the estimated number of genes in different organisms:
Species Common name length of DNA (kbp)* no of genes
phage λ virus 48 60
Eschericia coli Bacterium 4 639 4 000
Saccharomyces cerevisiae Yeast 13 500 6 000
Caenorhabditis elegans nematode worm 90 000 ~10 000
Drosophila melaogaster fruit fly 165 000 ~10 000
Homo sapiens Human 3 150 000 ~30 000
*
kbp = kilo base pairs, i.e. thousands of nucleotide monomers.
Amazingly, genes only seem to comprise about 2% of the DNA in a cell. The majority of the DNA
does not form genes and doesn’t seem to do anything. The purpose of this junk DNA remains a
mystery!

Replication - DNA Synthesis

DNA is copied, or replicated, before every cell division, so that one identical copy can go to each
daughter cell. The method of DNA replication is obvious from its structure: the double helix unzips
and two new strands are built up by complementary base-pairing onto the two old strands.
original DNA
1 double helix
enzyme unzips
2 DNA double helix
nucleotides base-pair to old
A T 4 strands with hydrogen bonds
C G
C G
T A T A
C G CG 5 DNA polymerase enzyme joins nucleotides
together with covalent phosphodiester bonds
G C GC
A T T G A T
A T T A A AT
C C
A
6 enzyme rewinds
3 DNA into double helix
free
nucleotides There are two kinds of bonds involved in DNA
replication:
Weak hydrogen bonds are formed between
bases and don't need an enzyme.
Strong covalent bonds are formed between
7 adjacent nucleotides in a strand. They are
two copies
made by the enzyme DNA polymerase.
of original DNA
1. Replication starts at a specific sequence on the DNA molecule called the replication origin.
2. An enzyme unwinds and separates the two strands of DNA, breaking the hydrogen bonds
between the base pairs.
3. The new DNA is built up from the four nucleotides (A, C, G and T) that are present in the
nucleoplasm.
4. These nucleotides attach themselves to the bases on the old strands by complementary base
pairing. Where there is a T base, only an A nucleotide will bind, and so on.
5. The enzyme DNA polymerase joins the new nucleotides to each other by strong covalent
phosphodiester bonds, forming the sugar-phosphate backbone. This enzyme is enormously
complex and contains 18 subunits.
6. Another enzyme winds the new strands up to form double helices.
7. The two new DNA molecules are identical to the old molecule. Each new DNA molecule
contains one "new" strand and one "old" strand.

DNA replication can takes a few hours, and in fact this limits the speed of cell division. One reason
bacteria can reproduce so fast is that they have a relatively small amount of DNA. In eukaryotes
replication is speeded up by taking place at thousands of sites along the DNA simultaneously.
replication forks
The Meselson-Stahl Experiment

This replication mechanism is sometimes called semi-conservative replication, because each new
DNA molecule contains one new strand and one old strand. This need not be the case, and
alternative theories suggested that a "photocopy" of the original DNA could be made, leaving the
original DNA conserved (conservative replication), or the old DNA molecule could be dispersed
randomly in the two copies (dispersive replication). The evidence for the semi-conservative method
came from an elegant experiment performed in 1958 by Matthew Meselson and Franklin Stahl.
They used the bacterium E. coli together with the technique of density gradient centrifugation,
which separates molecules on the basis of their density.
1. Grow bacteria light DNA

on medium purify DNA
with normal and centrifuge CsCl
14
NH4 solution These first two steps are a
calibration. They show that the
method can distinguish between
14
DNA containing N and that
2. Grow bacteria 15
containing N. The DNA is
for many purify DNA visualised under UV light.
generations
on medium and centrifuge
15
with NH4 heavy DNA
This is the crucial step. The DNA

14 14
3. Return to NH4 has replicated just once in N
purify DNA
medium for 20 intermediate medium. The resulting DNA is not
mins (one and centrifuge DNA heavy or light, but exactly half way
generation) between the two. This rules out
conservative replication.
14 light DNA After two generations the DNA is

4. Grow on NH4 purify DNA
intermediate either light or half-and-half. This
mediun for 40 rules out dispersive replication. The
mins (two and centrifuge DNA
results are all explained by semi-
generations) conservative replication.

RNA
RNA is a nucleic acid like DNA, but with 4 differences:
• RNA is made of ribose nucleotides instead of deoxyribose nucleotides

• RNA has the base uracil instead of thymine
• RNA is single stranded (though it can fold into 3-dimentional structures)
• RNA is shorter than DNA
There are three kinds of RNA, with three different jobs:
Messenger RNA (mRNA)

mRNA carries the "message" that codes for a particular protein from the nucleus (where the DNA
master copy is) to the cytoplasm (where proteins are synthesised). It is single stranded and just long
enough to contain one gene only. It has a short lifetime and is degraded soon after it is used.
Ribosomal RNA (rRNA)

rRNA, together with proteins, form ribosomes, which are the site of
mRNA translation and protein synthesis. Ribosomes have two large
25 nm subunit
subunits, small and large, and are assembled in the nucleolus of the
small
nucleus and exported into the cytoplasm. rRNA is coded for by subunit
numerous genes in many different chromosomes. Ribosomes free in the cytoplasm make proteins
for use in the cell, while those attached to the RER make proteins for export.
Transfer RNA (tRNA)

tRNA is an “adapter” that matches amino acids to their codon. tRNA amino
acid
is only about 80 nucleotides long, and it folds up by complementary A
C amino acid
base pairing to form a looped clover-leaf structure. At one end of the C attachment
site
molecule there is always the base sequence ACC, where the amino
acid binds. On the middle loop there is a triplet nucleotide sequence base pairing
called the anticodon. There are 64 different tRNA molecules, each
with a different anticodon sequence complementary to the 64 non-paired
loops
different codons. The amino acids are attached to their tRNA
molecule by specific aminoacyl tRNA synthase enzymes. These are
highly specific, so that each amino acid is attached to a tRNA anticodon
adapter with the appropriate anticodon.

Transcription - RNA Synthesis

DNA never leaves the nucleus, but proteins are synthesised in the cytoplasm, so a copy of each
gene is made to carry the “message” from the nucleus to the cytoplasm. This copy is mRNA, and
the process of copying is called transcription.
A C 2
G U
RNA polymerase
1
promoter 4 3
mRNA
5 mRNA for
one gene
6 nuclear pore
nuclear envelope
cytoplasm
rough endoplasmic reticulum ribosomes
1. The start of each gene on DNA is marked by a special sequence of bases called the promoter.
2. The RNA molecule is built up from the four ribose nucleotides (A, C, G and U) in the
nucleoplasm. The ribose nucleotides attach themselves to the bases on the DNA by
complementary base pairing, just as in DNA replication. However, only one strand of RNA is
made. The DNA strand that is copied is called the template strand. The other strand is a
complementary copy, called the non-template strand.
3. The new nucleotides are joined to each other by strong covalent phosphodiester bonds by the
enzyme RNA polymerase.
4. Only about 8 base pairs remain attached at a time, since the mRNA molecule peels off from the
DNA as it is made. A winding enzyme rewinds the DNA.
5. At the end of the gene the transcription stops, so the mRNA molecule is just the length of the
gene.
6. The mRNA diffuses out of the nucleus through a nuclear pore into the cytoplasm. There, it
attaches to ribosomes for translation. It usually doesn't have far to go to find a ribosome, as many
are attached to the rough endoplasmic reticulum, which is contiguous with the nuclear envelope.

Introns and Exons

It turns out that genes contain many regions that are not needed as part of the protein code. These
are called introns (for interruption sequences), while the parts that are needed are called exons (for
expressed sequences). All eukaryotic genes have introns, and they are usually longer than the exons,
so genes are often much longer than they really need to be! No one knows what these introns are
for, but they need to be removed before the mRNA can be translated into protein.
exon 1 exon 2 exon 3 exon 4 exon 5

1 intron intron intron intron
primary
transcript
introns
2 introns cut out exons
1 2 3 4 5
+ introns
mature mRNA
3 – just exons
1. The initial mRNA that is transcribed, or primary transcript, is an exact copy of the gene on the
DNA, so it contain exons and introns.
2. The introns in the mRNA are cut out and the exons are joined together by enzymes. Some of this
joining (or splicing) is done by the RNA intron itself, acting as an RNA enzyme. The recent
discovery of these RNA enzymes, or ribozymes, illustrates what a diverse and important
molecule RNA is. Other splicing is performed by RNA/protein complexes called snurps.
3. The result is a shorter mature RNA containing only exons. The introns are broken down.

The Genetic Code

The sequence of bases on DNA codes for the sequence of amino acids in proteins. But there are 20
different amino acids and only 4 different bases, so the bases are read in groups of three. This gives
43 or 64 combinations, more than enough to code for 20 amino acids. A group of three bases coding
for an amino acid is called a codon, and the meaning of each of the 64 codons is called the genetic
code.
The Genetic Code (mRNA codons)

UUU CUU AUU GUU
phe
UUC CUC AUC ile GUC
leu val
UUA CUA AUA GUA
leu
UUG CUG AUG start/met GUG
UCU CCU ACU GCU
UCC CCC ACC GCC
ser pro thr ala
UCA CCA ACA GCA
UCG CCG ACG GCG
UAU CAU AAU GAU
tyr his asn asp
UAC CAC AAC GAC
UAA CAA AAA GAA
stop gln lys glu
UAG CAG AAG GAG
UGU CGU AGU GGU
cys ser
UGC CGC AGC GGC
arg gly
UGA stop CGA AGA GGA
arg
UGG trp CGG AGG GGG
There are several interesting points from this code:
• The code is degenerate, i.e. there is often more than one codon for an amino acid.
• The degeneracy is on the third base of the codon, which is therefore less important than the
others.
• One codon means "start" i.e. the start of the gene sequence. It is AUG, which also codes for
methionine. Thus all proteins start with methionine (although it may be removed later). AUG in
the middle of a gene simply codes for methionine.
• Three codons mean "stop" i.e. the end of the gene sequence. They do not code for amino acids.
• The code is read from the 5' to 3' end of the mRNA, and the protein is made from the N to C
terminus ends.

Translation - Protein Synthesis

ribosome
initiation
codon
1. A ribosome attaches to the mRNA at an initiation
codon (AUG). The ribosome encloses two
CA
G U G C A U G C U G U G C mRN A
AAC
codons. UU
codons
met
2. The first tRNA molecule with an amino acid
attached (met-tRNA) diffuses to the ribosome. Its anticodon
CA UA C
anticodon attaches to the first mRNA codon by GUG
CA UGCUGUGCA AC
UU
codon
complementary base pairing.
3. The next amino acid-tRNA attaches to the met leu
adjacent mRNA codon (CUG, leu in this case) by

complementary base pairing. CA
GUG
UA CGA C
CA UGCUGUGCA AC
UU
peptide bond
4. The bond between the amino acid and the tRNA met leu
cut
is cut and a peptide bond is formed between the
two amino acids. These operations are catalysed CA
GUG
UA CGA C
CA UGCUGUGCA AC
UU
by enzymes in the ribosome called ribozymes.
5. The ribosome moves along one codon so that a met leu cys
new amino acid-tRNA can attach. The free tRNA

U G GA CA CG
molecule leaves to collect another amino acid. CAU GCUGU
G CA ACUUA
C C
The cycle repeats from step 3.
6. The polypeptide chain elongates one amino acid

ala stop codon
at a time, and peels away from the ribosome, cys
val phe
folding up into a protein as it goes. This continues
for hundreds of amino acids until a stop codon is UG A A G
C G UC UU CUAGC CA
AG GG
reached, when the ribosome falls apart, releasing G
the finished protein.

A single piece of mRNA can be translated by many ribosomes simultaneously, so many protein
molecules can be made from one mRNA molecule. A group of ribosomes all attached to one piece
of mRNA is called a polyribosome, or a polysome.
finished
growing polypeptide chain
protein
A
mRN
Post-Translational Modification
In eukaryotes, proteins often need to be altered before they become fully functional. Because this
happens after translation, it is called post-translational modification. Modifications are carried out
by other enzymes and include: chain cutting, adding methyl or phosphate groups to amino acids,
adding sugars (to make glycoproteins) or lipids (to make lipoproteins).
Regulation of Gene Expression

Not all genes make proteins. Some important genes control the expression of other genes, and so are
called control genes. Control genes usually work by regulating transcription, so mRNA is only
made in a cell where it is needed and when it is needed. Remember, each cell in your body contains
all of your genes, but only a few are actually expressed. For example skin cells could make amylase
but don't, and kidney cells could make haemoglobin, but don't. This is because of control genes.
Control genes are if anything even more important than structural genes in controlling
characteristics. For example control genes control the development of an embryo and determine
which cells differentiate into which kind of tissue. They also control the timing of events such as
puberty, flowering or ageing. So most characteristics are controlled by many genes working
together, and most genes affect many different aspects of a cell’s function. Characteristics are also
influenced by non-genetic factors, such as diet and environment.
Some genes (called oncogenes) control cell division and growth, and it is a malfunction in these
genes that causes cancer. The regulation of gene expression is a highly complex subject, and is still
poorly understood.

Mutations
Mutations are changes in genes, which are passed on to daughter cells. DNA is a very stable
molecule, and it doesn't suddenly change without reason, but bases can change when DNA is being
replicated. Normally replication is extremely accurate, and there are even error-checking procedures
in place to ensure accuracy, but very occasionally mistakes do occur (such as a T-C base pair). So a
mutation is a base-pairing error during DNA replication.
A change in a gene could cause a change in the protein made by the gene, and so a change in the
cell function:
change change change different change in change in change in change
in base in base in mRNA amino acid amino acid protein protein in cell
of DNA of mRNA codon in protein sequence structure function function
Many of the proteins in cells are enzymes, and most changes in enzymes will stop them working
(because there are far more ways of making an inactive enzyme than there are of making a working
one). When an enzyme stops working, a metabolic block can occur, when a reaction in a cell doesn't
happen, so the cell's function is changed.
gene P gene Q
Compound A enzyme P Compound B enzyme Q Compound C

In this example of a metabolic pathway two enzymes (P and Q) are needed to make compound C
from compound A. If a mutation occurs in gene P then enzyme P won't be made (or at least will be
the wrong shape so won't work), so compound B can't be made. And with no compound B then
compound C cannot be made, even if enzyme Q is functional. It's just possible (though unlikely)
that a mutation in gene P could make a modified enzyme P that actually worked faster than the
original enzyme. This means the metabolic pathway could be faster and the cell's function could be
improved.
Since mutations change genes, they give rise to new alleles (i.e. different versions of genes). A cell
with the original, functional gene has one allele, while a cell with a mutated, non-functional version
of the same gene has a different allele. In the above example if compound A was a white pigment in
a flower and compound C was a red pigment, then the "red" allele for flower colour would be the
original gene P (and functional enzyme P) while the "white" allele for flower colour would be a
mutated gene P (and non-functional enzyme P).

So there are three possible phenotypic effects of a mutation:

• Most mutations have no phenotypic effect because they don't change the protein or they are not
expressed in this cell. These are called silent mutations, and we all have a few of these.
• Of the mutations that have a phenotypic effect, most will have a deleterious effect.
• Very rarely a mutation can have a beneficial phenotypic effect, such as making an enzyme work
faster, or a structural protein stronger, or a receptor protein more sensitive. A small mutation in a
control gene can have a very large phenotypic effect, such as developing extra limbs or
flowering more often. Although rare, these beneficial mutations are important as they drive
evolution. Examples include modified enzymes that make bacteria resistant to antibiotics, cows
that produce milk constantly, sweetcorn that tastes sweet and almonds that aren't poisonous.
There are three kinds of gene mutation, shown in this diagram:
mRNA . . . C G C G U U U C C . . . (part of original gene)

protein arg val ser
G A G +A
SUBSTITUTION DELETION INSERTION

C G C A U U U C C C G C U U U C C A C G C A G U U U C
arg ile ser arg phe pro arg ser phe
Only one amino acid Reading frame altered. Reading frame altered.
altered. Rest of protein OK. Rest of protein wrong. Rest of protein wrong.
• Substitution mutations only affect one amino acid, so tend to have less severe effects. In fact if
the substitution is on the third base of a codon it may have no effect at all, because the third base
often doesn't affect the amino acid coded for (e.g. all codons beginning with CC code for
proline). However, if a mutation leads to a premature stop codon the protein will be incomplete
and certainly non-functional.
• Deletion and insertion mutations have more serious effects because they are frame shift
mutations i.e. they change the codon reading frame even though they don't change the actual
sequence of bases. So all amino acids "downstream" of the mutation are wrong, and the protein
is completely wrong and non-functional. However, the effect of a deletion can be cancelled out
by a near-by insertion, or by two more deletions, because these will restore the reading frame. A
similar argument holds for a substitution.

Mutation Rates and Mutagens

Mutations are normally very rare, which is why members of a species all look alike and can
interbreed. However the rate of mutations is increased by chemicals or by radiation. These are
called mutagenic agents or mutagens, and include:
• High-energy ionising radiation such as x-rays, ultraviolet rays, α, β, or γ rays from radioactive
sources. This ionises the bases so that they don't form the correct base pairs. Note that low-
energy radiation (such as visible light, microwaves and radio waves) doesn't have enough energy
to affect DNA and so is harmless.
• Intercalating chemicals such as mustard gas (used in World War 1), which bind to DNA
separating the two strands.
• Chemicals that react with the DNA bases such as benzene, nitrous acid, and tar in cigarette
smoke.
• Viruses. Some viruses can change the base sequence in DNA causing genetic disease and cancer.
During the Earth's early history there were far more of these mutagens than there are now, so the
mutation rate would have been much higher than now, leading to a greater diversity of life. Some of
these mutagens are used today in research, to kill microbes or in warfare. They are often
carcinogens since a common result of a mutation is cancer.

DNA and Chromosomes

The DNA molecule in a single human cell is 1 m long, so is 10 000 times longer than the cell in
which it resides (< 100µm). (Since an adult human has about 1014 cells, all the DNA is one human
would stretch about 1014 m, which is a thousand times the distance between the Earth and the Sun.)
In order to fit into the cell the DNA is cut into shorter lengths and each length is tightly wrapped up
with histone proteins to form a complex called chromatin. During most of the life of a cell the
chromatin is dispersed throughout the nucleus and cannot be seen with a light microscope. At
various times parts of the chromatin will unwind so that genes on the DNA can be transcribed. This
allows the proteins that the cell needs to be made.
Just before cell division the DNA is replicated, and more histone proteins are synthesised, so there
is temporarily twice the normal amount of chromatin. Following replication the chromatin then
coils up even tighter to form short fat bundles called chromosomes. These are about 100 000 times
shorter than fully stretched DNA, and therefore 100 000 times thicker, so are thick enough to be
seen with the light microscope. Each chromosome is roughly X-shaped because it contains two
replicated copies of the DNA. The two arms of the X are therefore identical. They are called
chromatids, and are joined at the centromere. (Do not confuse the two chromatids with the two
strands of DNA.) The complex folding of DNA into chromosomes is shown below.
chromosome
one chromatid
chromatin
histone proteins centromere
DNA double helix

micrograph of a single chromosome

Karyotypes and Homologous Chromosomes

If a dividing cell is stained with a special fluorescent dye and examined under a microscope during
cell division, the individual chromosomes can be distinguished. They can then be photographed and
studied. This is a difficult and skilled procedure, and it often helps if the chromosomes are cut out
and arranged in order of size.
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21 22 XY
This display is called a karyotype, and it shows several features:

• Different species have different number of chromosomes, but all members of the same species
have the same number. Humans have 46 (this was not known until 1956), chickens have 78,
goldfish have 94, fruit flies have 8, potatoes have 48, onions have 16, and so on. The number of
chromosomes does not appear to be related to the number of genes or amount of DNA.
• Each chromosome has a characteristic size, shape and banding pattern, which allows it to be
identified and numbered. This is always the same within a species. The chromosomes are
numbered from largest to smallest.
• Chromosomes come in pairs, with the same size, shape and banding pattern, called homologous
pairs ("same shaped"). So there are two chromosome number 1s, two chromosome number 2s,
etc, and humans really have 23 pairs of chromosomes.
• One pair of chromosomes is different in males and females. These are called the sex
chromosomes, and are non-homologous in one of the sexes. In humans the sex chromosomes are
homologous in females (XX) and non-homologous in males (XY). In other species it is the other
way round. The non-sex chromosomes are called autosomes, so humans have 22 pairs of
autosomes, and 1 pair of sex chromosomes.

It is important to understand exactly what homologous chromosomes are. We have two copies of
each chromosome because we inherit one copy from each parent, so each homologous pair consists
of a maternal and paternal version of the same chromosome. Since the homologous chromosomes
contain the same genes, this also means we have two copies of each gene (again, one from each
parent). This is why we write two letters for each gene in a genetic cross. The two homologous
chromosomes may have the same versions (or alleles) of the gene (e.g. AA), or they may have
different alleles, because one copy is a mutation (Aa).
Sometimes the chromosomes in a cell nucleus are represented by

rods called ideograms, although these structures never actually exist round
seeds
round
seeds
because the chromatin is usually uncoiled. Each ideogram

represents the long coiled DNA molecule in one chromosome. This
diagram shows a pair of homologous chromosomes with two genes
purple white
flowers flowers
marked. The plant cell containing these chromosomes is
homozygous for the seed shape gene (RR) and heterozygous for the
flower colour gene (Pp). maternal paternal
chromosome chromosome
The only time chromosomes can actually be seen is during cell

division. At this point in the cell cycle each chromosome is made of round round round round
seeds seeds seeds seeds
two identical chromatids, because each DNA molecule has now

been replicated. This diagram shows the same pair of homologous
chromosomes during mitosis. The two chromatids in each
purple purple white white
flowers flowers flowers flowers
chromosome contain the same alleles because they're exact replicas
of each other. But again the two homologous chromosome contain
the same genes but different alleles. maternal paternal
chromosome chromosome
Chromatin DNA + histone complex during interphase
Chromosome compact X-shaped form of chromatin formed (and visible) during mitosis
the two arms of an X-shaped chromosome. The two chromatids are identical since
Chromatids
they are formed by DNA replication.
Homologous two chromosome of the same size and shape, one originating from each parent.
chromosomes They contain the same genes, but different alleles.

Gene Loci
Since the DNA molecule extends form one end of a chromosome to the other, and the genes are
distributed along the DNA, then each gene has a defined position on a chromosome. This position is
called the locus of the gene, and the loci of thousands of human genes are now known. There are on
average about 1 000 genes per chromosome, although of course the larger chromosomes have more
than this, and the smaller ones have fewer. This diagram shows the loci of a very few example
genes in humans:
r
be
nd
um
ou
)
es
en
sf
s
ba
ne
om
(M
os
ge
of
th
m
ro Sample Genes
ng
ch
N
le
1 246 2610 Elastase (protease); Amylase; Skeletal muscle actin

2 240 1748 Lactase; Glucagon
3 193 1381 Alkaptonuria
4 191 1024 Huntingtin
5 181 1190 Asthma
6 170 1394 Antibodies; Potassium channel
7 157 1378 CFTR; Trypsin (endopeptidase)
8 143 927
9 132 1076 Red blood cell antigens (blood groups)
10 135 983 Smooth muscle actin; Lipase
11 137 1692 Insulin; Haemoglobin
12 132 1268 HOX genes (embryo development)
13 113 496 Breast cancer; skeletal muscle myosin
14 104 1173 AAT
15 99 906 Cardiac muscle actin; Tay-Sachs disease
16 81 1032 Calcium pump in fast skeletal muscle
17 80 1394 Human Growth Hormone
18 77 400 Leukemia
19 60 1592 Alzheimers
20 63 710 SCID
21 45 337 Enterokinase (endopeptidase); Down syndrome
22 48 701
X 148 1141 Rhodopsin (retina photoreceptor); Blood clotting factor VIII
Y 59 255 SRY (sex-determining genes)
mt 0.02 37 Respiration enzymes

The Cell Cycle

Cells are not static structures, but are created and die. The life of a cell is called the cell cycle and
has two main phases:
1. Interphase 2. Mitotic Phase

mi to
Genes are expressed into proteins, and s is Cell division, or mitosis, takes
the cell does its thing. Can last from place. The cell cycle starts again
minutes to years. Towards the end of for each daughter cell.
interphase the DNA, histones and other
proteins are replicated, so there is
temporarily twice the normal amount of
DNA.
i nt er
phase
In different cell types the cell cycle can last from hours to years. For example bacterial cells can
divide every 30 minutes under suitable conditions, skin cells divide about every 12 hours on
average, liver cells every 2 years, and muscle cells never divide at all after maturing, so remain in
the growth phase for decades.
• Interphase can be sub-divided into growth and synthesis phases. In the growth phase the cell
grows and does whatever it does (e.g. respires, synthesises molecules, secretes hormones,
contracts, transmits nerve impulses, etc.). In the synthesis phase DNA and histones are
replicated in preparation for mitosis.
• The mitotic phase can be sub-divided into four phases (prophase, metaphase, anaphase and
telophase). Mitosis is strictly nuclear division, and is followed by cytoplasmic division, or
cytokinesis, to complete cell division. Mitosis results in two “daughter cells”, which are
genetically identical to each other, and is used for growth and asexual reproduction. The details
of each of these phases are shown on the next page.

Cell Division by Mitosis

This cell has n = 2; i.e. 2 pairs of homologous chromosomes.
centrioles
Interphase • no chromosomes visible
chromatin
nucleolus • DNA, histones and centrioles all replicated
nuclear
envelope
cell
membrane
Prophase • chromosomes condensed and visible

• centrioles at opposite poles of cell
• nucleolus disappears
Metaphase • nuclear envelope disappears

• chromosomes align along equator of cell
• spindle fibres (microtubules) connect centrioles to
chromosomes
Anaphase • centromeres split, allowing chromatids to separate

• chromatids move towards poles, centromeres first,
pulled by motor proteins "walking" along the
microtubule tracks
Telophase • spindle fibres disperse

• nuclear envelopes form
• chromosomes uncoil and become invisible
Cytokinesis • In animal cells a ring of actin filaments forms round

the equator of the cell, and then tightens to form a
cleavage furrow, which splits the cell in two.
• In plant cells vesicles move to the equator, line up

and fuse to form two membranes called the cell
plate. A new cell wall is laid down between the
membranes, which fuses with the existing cell wall.

Asexual Reproduction
Asexual reproduction is the production of offspring from a single parent using mitosis. The
offspring are therefore genetically identical to each other and to their “parent”- in other words they
are clones. A clone is defined as a cell or organism (or even a molecule of DNA) that is genetically
identical to another cell or organism (or molecule of DNA). Asexual reproduction is very common
in nature, and in addition we humans have developed some new, artificial methods. The Latin terms
in vivo (“in life”, i.e. in a living organism) and in vitro (“in glass”, i.e. in a test tube) are often used
to describe natural and artificial techniques respectively. Cloning (both natural and artificial) is of
great commercial importance as brewers, pharmaceutical companies, farmers and plant growers all
want to be able to reproduce “good” organisms exactly. Natural methods of asexual reproduction
are often quite suitable for some organisms (such as yeast, potatoes and strawberries), but many
important plants and animals do not reproduce asexually (such as apples, bananas or sheep), so
artificial methods have to be used.
Some different methods of asexual reproduction are summarised in this table.

Methods of Asexual Reproduction
Natural Methods Artificial Methods
binary fission
cell culture
Microbes budding
fermenters
fragmentation
cuttings
vegetative propagation
Plants grafting
parthenogenesis
micropropagation
budding
 invertebrates  embryo splitting  any 
Animals fragmentation  only   animal 
parthenogenesis   somatic cell cloning  
Cloning Plants
The natural methods of asexual reproduction used by plants are referred to as vegetative
propagation. A bud grows from a vegetative (i.e. not reproductive) part of the plant (usually the
stem) and develops into a complete new plant, which eventually becomes detached from the parent
plant. There are numerous forms of vegetative reproduction, including:
• bulbs (e.g. onion, daffodil) • runners (e.g. strawberry, creeping buttercup)
• corms (e.g. crocus, gladiolus) • tubers (e.g. potato, dahlia)
• rhizomes (e.g. iris, couch grass) • tap roots (e.g. carrot, turnip)
• stolons (e.g. blackberry, bramble) • tillers (e.g. grasses)

Many of these methods are also perenating organs, which means they contain a food store and are
used for survival over winter as well as for asexual reproduction. Since vegetative reproduction
relies entirely on mitosis, all offspring are clones of the parent.
parent
plant
scale leaves clones of parent
food store
runner
new shoot
stem
roots
Bulb Runners Tubers
There are three artificial methods used to clone plants: cuttings, grafting and micropropagation.
Cuttings. This is a very old method of cloning plants.

Parts of a plant stem (or even leaves) are cut off and
simply replanted in wet soil. Each cutting produces roots
and grows into a complete new plant, so the original cut
plant can be cloned many times. Rooting is helped if the
cuttings are dipped in rooting hormone (auxin). Many
flowering plants, such as geraniums, African violet and rooting
hormone
chrysanthemums are reproduced commercially by
cuttings.
Grafting. This is another ancient technique, used for plant

species that cannot grow roots from cuttings. Instead they
can often be cloned by grafting a stem cutting (called a
scion
scion) onto the lower part of an existing plant (called the
rootstock). One rootstock can take several scions, and need
+
not even be the same species as the scion. The resulting root
stock binding
hybrid will produce the flowers and fruits of the scion, but
its size and hardiness will be determined by the rootstock.
Careful selection of rootstock species can result in plants
that are easier to harvest or can grow in particular soils.
Almost all fruit trees, such as apples and pears, are clones of
a few popular varieties grafted onto hardy rootstock.

Tissue Culture (or micropropagation). This is a more modern, and very efficient, way of cloning
plants. Small samples of plant tissue, called an explant, can be grown on agar plates in the
laboratory in much the same way that bacteria can be grown. Any plant tissue can be used for this
(e.g. from a leaf). The plant tissue can be separated into individual cells, each of which can grow
into a mass of undifferentiated cells called a callus. If the correct plant hormones are added these
cells can develop into whole plantlets, which can eventually be planted outside, where they will
grow into normal-sized plants. Conditions must be kept sterile to prevent infection by microbes.
leaf cells explant shoot root

stimulating stimulating plant
hormones hormones out
callus culture
nutrient agar plantlet normal plant
Micropropagation is used on a large scale for fruit trees, ornamental plants and plantation crops
such as oil palm, date palm, sugar cane and banana. The advantages are:
• thousands of clones of a particularly good plant can be made quickly and in a small space
• the technique works for plants species that cannot be asexually propagated by other means, such
as palms and bananas.
• disease-free plants can be grown from a few disease-free cells. In the field, almost all crop plants
are infected with viruses.
• a single cell can be genetically modified and turned into many identical plants
Cloning Animals
No vertebrate animal can reproduce naturally by asexual reproduction, and so it has proved very
difficult to develop artificial methods of cloning animals. The problem is that in vertebrates (unlike
plants and some invertebrates) the differentiation process cannot be reversed. So a skin cell cannot
be turned into a liver cell or heart cell. A lot of research is going into finding stem cells –
reasonably undifferentiated animal cells that can develop into different tissues. Although some
animal cells can be grown in culture, they cannot therefore be grown into complete animals, so
tissue culture cannot be used for cloning animals. Two techniques for cloning vertebrates have been
developed to circumvent these problems:
Embryo Cloning (or Embryo Splitting). The most effective technique for cloning animals is to
duplicate embryo cells before they have irreversibly differentiated into tissues. It is difficult and
quite expensive, so is only worth it for commercially-important farm animals, such as prize cows, or

genetically engineered animals. A female animal is given a fertility drug (FSH) so that she produces
many mature eggs (superovulation). The eggs are then surgically removed from the female’s
ovaries. The eggs are fertilised in vitro (IVF) using selected sperm from a prize male. The fertilised
eggs (zygotes) are allowed to develop in vitro for a few days until the embryo is at the 16-cell stage.
This young embryo can be split into 16 individual cells, which will each develop again into an
embryo. (This is similar to the natural process when a young embryo splits to form identical twins.)
The identical embryos can then be transplanted into the uterus of surrogate mothers, where they will
develop and be born normally. These animals are clones of each other but not of their parents, since
the zygote was made by sexual reproduction.
Could humans be cloned this way? Almost certainly yes. Human embryos have been split and
cloned to the stage of a few cells, for example to make stem cells for treating diseases. These
therapeutic cloning experiments with human embryos are permitted, but are very tightly controlled.
Growing cloned human babies (reproductive cloning) is not permitted in most countries (including
the UK) for ethical reasons.
Somatic Cell Cloning (or Nuclear Transfer). The problem with embryo cloning is that you don’t
know the characteristics of the animal you are cloning. By selecting good parents you hope it will
have good characteristics, but you will not know until the animal has grown. It would be far better
to clone a mature animal, whose characteristics you know. Until recently it was thought impossible
to grow a new animal from the somatic cells of an existing vertebrate animal. However, techniques
have gradually been developed to do this, first with frogs in the 1970s, then with sheep (the famous
“Dolly”) in 1996 and more recently with monkeys in 2001.
The technique used to create Dolly is similar to embryo cloning, but has one crucial difference. The
cells used for Dolly were from the skin of the udder of an adult sheep, so were fully differentiated
somatic cells. They were gown in tissue culture for several years before they were used. One cell
was fused with a unfertilised egg cell which had had its nucleus removed. This combination of a
diploid nucleus in an unfertilised egg cell was a bit like a zygote, and sure enough it developed into
an embryo. The embryo was implanted into the uterus of a surrogate mother, and developed into an
apparently normal sheep, Dolly. It took 277 attempts to achieve success with Dolly, but once the
technique is improved it may be possible to combine this technique with embryo cloning to make
many clones of an adult animal. Dolly’s “mother” (identical twin?) was just an ordinary sheep, but

in the future prize animals (or genetically engineered ones) could be cloned in this way. Dolly died
in 2003 aged 6 after leading a fairly normal life and giving birth to healthy lambs of her own.
Embryo Cloning Somatic Cell Cloning

(embryo splitting) (nuclear transfer)
A B
Select prize cow. scrape cells Give FSH to superovulate

Give FSH to superovulate Select prize bull from udder tissue
collect eggs
collect eggs collect sperm
somatic cell remove nucleus

in vitro fertilisation (could be grown in
culture for years)
holding
pipette sucking
pipette
fuse cells with electric current
Grow in vitro to 16-cell embryo

somatic egg cell
cell
Grow in vitro to 16-cell embryo
split embryo into several "identical twins"
implant into surrogate mother

grow to 16-cell stage & implant into surrogate mothers
each calf is a clone lamb is a clone of sheep A

Sexual Reproduction
Sexual reproduction is the production of offspring from two parents using gametes. The cells of the
offspring therefore have two sets of chromosomes, one from each parent. Sexual reproduction
involves two stages:
• Meiosis – the special cell division that halves the chromosome number from the normal, diploid
number (2n) to the haploid number (n).
• Fertilisation – the fusion of two haploid gametes to form a diploid zygote
Meiosis
Meiosis is a form of cell division. It starts with DNA replication, like mitosis, but then proceeds
with two divisions one immediately after the other. Meiosis therefore results in four daughter cells
rather than the two cells formed by mitosis. It differs from mitosis in two important aspects:
• In meiosis the chromosome number is halved from the diploid number to the haploid number.
This is necessary so that the chromosome number remains constant from generation to
generation. The halving is done in a particular way: meiosis ensures that each haploid cell has
one of each homologous pair of chromosomes. So for example human gametes have 23
chromosomes: one of each homologous pair. Remember that other species have different haploid
numbers.
meiosis
Diploid cell 4 haploid cells

2 copies of each chromosome 1 copy of each chromosome
• In meiosis the chromosomes are re-arranged during meiosis to form new combinations of genes.
This genetic recombination is vitally important and is a major source of genetic variation. It
means for example that of all the millions of sperm produced by a single human male, the
probability is that no two will be identical.
You don’t need to know the details of meiosis at this stage (that comes in module 4).

Gametes
Gametes are the haploid sex cells that will fuse together to form a new diploid individual. Gametes
may not be made directly by meiosis, but instead by one or more mitotic divisions of haploid cells.
In all plants and animals there are two kinds of gametes – female and male.
Female gametes Male gametes

Female gametes (ova or eggs in animals, ovules Male gametes are small cells that can move. If
in plants) are relatively large cells and they tend they can propel themselves they are called
to be stationary. They contain food reserves motile (e.g. animal sperm) but if they can easily
(lipids, proteins, carbohydrates) to nourish the be carried by the wind or animals they are called
embryo after fertilisation, and they are produced mobile (e.g. plant pollen). They are produced in
in fairly small numbers. Human females for very large numbers. Human males for example
example release about 500 ova in a lifetime. release about 108 sperm in one ejaculation.
Female: Few, Fixed, Food Male: Many, Mobile, Minute
It is this difference in gametes that actually defines the sex of an individual. Those individuals that
produce small mobile gametes are the males, and those that produce the larger gametes are the
females. In some species (such as most flowering plants) the same individual organisms can
produce both male and female gametes, so they do not have distinct sexes and are called
hermaphrodites. In other species (such as mammals) there are two distinct sexes, each producing
their own gametes. These organisms are called unisexual.
These diagrams of human gametes illustrate the differences between male and female gametes.
follicle cells acrosome

jelly coat head nucleus
membrane mid- mitochondria
cytoplasm piece membrane
100µm nucleus
flagellum
nucleolus
polar body tail
lipid droplets 10µm
A Human Ovum A Human Sperm

Fertilisation
Fertilisation is the fusion of two gametes to form a zygote.
In humans this takes place near the top of the oviduct. Hundreds
of sperm reach the egg and use their flagella to swim through the
follicle cells (shown in this photo). When they reach the jelly coat
surrounding the ovum they bind to receptors and this stimulates
the rupture of the acrosome membrane in the sperms, releasing
digestive enzymes, which digest a path through the jelly coat.
When a sperm reaches the ovum cell the two membranes fuse and
the sperm nucleus enters the cytoplasm of the ovum. This triggers
a series of reactions in the ovum (called the cortical reaction) that cause the jelly coat to thicken and
harden, preventing any other sperm from entering the ovum. The sperm and egg nuclei then fuse,
forming a diploid zygote.
In plants fertilisation takes place in the ovary at the base of the carpel. The haploid male nuclei
travel down the pollen tube from the pollen grain on the stigma to the ovules in the ovary. In the
ovule two fusions between male and female nuclei take place: one forms the zygote (which will
become the embryo) while the other forms the endosperm (which will become the food store in the
seed). This double fertilisation is unique to flowering plants.
The Reason for Sex

The reason for sex is variation. For most of the history of life on Earth, organisms have reproduced
only by asexual reproduction. Each individual was a genetic copy (or clone) of its “parent”, and the
only variation was due to random genetic mutation. The development of sexual reproduction in the
eukaryotes around one billion years ago led to much greater variation and diversity of life. Sexual
reproduction is slower and more complex than asexual, but it has the great advantage of introducing
genetic variation (due to genetic recombination in meiosis and random fertilisation). This variation
allows species to adapt to their environment and so to evolve. This variation is clearly such an
advantage that practically all species can reproduce sexually. Some organisms can do both, using
sexual reproduction for genetic variety and asexual reproduction to survive harsh times.

Sexual Life Cycles

The stages of sexual reproduction can be illustrated by a sexual life cycle:
All sexually-reproducing species have the basic life cycle shown on diploid
cells
the right, alternating between diploid and haploid forms. In
addition, they will also use mitosis to grow into adult organisms, fertilisation meiosis
but the details vary with different organisms.
haploid
cells
In vertebrate animals (including humans), and in flowering plants mitosis

diploid zygote diploid adult
the dominant, long-lived adult form is diploid, and the haploid
gamete cells are only formed briefly. fertilisation meiosis
haploid
gametes
diploid
In the fungi kingdom the dominant, long-lived adult form is zygote
haploid. Haploid spores undergo mitosis and grow into complete,

differentiated adults (including large structures like mushrooms). At fertilisation meiosis
some stage two of these haploid cells fuse to form a diploid zygote, haploid adult haploid spores
which immediately undergoes meiosis to re-establish the haploid mitosis
state and complete the cycle.
In some plants and some invertebrate animals the life cycle shows mitosis
diploid zygote diploid adult

alternation of generations. These organisms have two distinct adult
forms; one diploid and the other haploid. In the simpler plants fertilisation meiosis
(mosses and liverworts) the haploid form is larger than the diploid haploid adult haploid spores
form, while in the higher plants (ferns and conifers) and animals the mitosis
diploid form is larger.

Genetic Engineering
Genetic engineering, also known as recombinant DNA technology, means altering the genes in a
living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various
kinds of genetic modification are possible:
• inserting a foreign gene from one species into another, forming a transgenic organism
• altering an existing gene so that its product is changed
• changing gene expression so that it is translated more (overexpressed), or less (deactivated).
Techniques of Genetic Engineering

Genetic engineering is a very young discipline, and is only possible due to the development of
techniques from the 1960s onwards. These techniques have been made possible from our greater
understanding of DNA and how it functions, following the discovery of its structure by Watson and
Crick in 1953. Although the final goal of genetic engineering is usually the expression of a gene in
a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and
then cloning it. This table lists the techniques that we shall look at in detail.
Technique Purpose Type

1 PCR To amplify very small samples of DNA
Analysing
2 Electrophoresis To separate fragments of DNA
DNA
3 DNA Sequencing To read the base sequence of a length of DNA
4 Restriction Enzymes To cut DNA at specific points, making small fragments
Manipulating
5 DNA Ligase To join DNA fragments together
DNA
6 Plasmids To carry DNA into cells and ensure replication
7 Transformation To deliver a gene into a living cell
8 Genetic Markers To identify cells that have been transformed Manipulating
9 Replica Plating To make exact copies of bacterial colonies on an agar plate Cells
10 Fermenters To grow large quantities of a microbe
The use of these techniques in a typical genetic engineering project (the manufacture of genetically-
engineered insulin by bacteria) is shown on the next page:

Extract
human 6 Extract
DNA bacterial
plasmids
or
cut out human cut plasmids
gene with with same Buy
Restriction 4 4 Restriction bacterial
or Enzyme Enzyme plasmids
1
Chemistry
mix
together
Amplify small
DNA sample
Sticky ends anneal. Several different products formed

5 Add DNA ligase
Sequence to join DNA backbone
human hybrid original circularised
gene plasmid plasmid human DNA
2 3
Insert plasmids into Bacterial cells

7 in culture flask
bacterial cells
Different bacterial
cells formed
no plasmid hybrid plasmid original plasmid human DNA only
8
Grow on agar
plates with killed resistant resistant killed
antibiotic 1
Grow on replica
Cells
agar plates with Identify

hybrid killed resistant
antibiotic 2
colonies
9
Grow transformed
bacteria in culture
flask in lab
10
Grow bacteria in industrial-scale Purify insulin from
fermenter. Genetically-modified medium and sell
bacteria synthesise insulin

1. Polymerase Chain Reaction (PCR)

The polymerase chain reaction is a technique used to copy (or amplify) DNA samples as small as a
single molecule. It was developed in 1983 by Kary Mullis, for which discovery he won the Nobel
Prize in 1993. PCR is simply DNA replication in a test tube. If a length of DNA is mixed with the
four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will
be replicated many times. The details are shown in this diagram:
DNA polymerase 4 nucleotides
1
Original DNA
2 Heat to 95°C
Strands separate
Add primers 3 Cool to 40°C

Repeat
Target sequence
5
Primers anneal
4 Heat to 72°C
DNA replicated
1. Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme
DNA polymerase.
2. Heat to 95°C for two minutes to breaks the hydrogen bonds between the base pairs and separate
the two strands of DNA. Normally (in vivo) the DNA double helix would be separated by an
enzyme.
3. Add primers to the mixture and cool to 40°C. Primers are short lengths of single-stranded DNA
(about 20 bp long) that anneal to complementary sequences on the two DNA strands forming
short lengths of double-stranded DNA. The DNA is cooled to 40°C to allow the hydrogen bonds
to form. There are two reasons for making short lengths of double-stranded DNA:
• The enzyme DNA polymerase requires some existing double stranded DNA to get it started.
• Only the DNA between the primer sequences is replicated, so by choosing appropriate
primers you can ensure that only a specific target sequence is copied.
4. The DNA polymerase enzyme can now build new stands alongside each old strand to make
double-stranded DNA. Each new nucleotide binds to the old strand by complementary base
pairing and is joined to the growing chain by a phosphodiester bond. The enzyme used in PCR is
derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot

springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its
optimum temperature is about 72°C, so the mixture is heated to this temperature for a few
minutes to allow replication to take place as quickly as possible.
5. Each original DNA molecule has now been replicated to form two molecules. The cycle is
repeated from step 2 and each time the number of DNA molecules doubles. This is why it is
called a chain reaction, since the number of molecules increases exponentially, like an explosive
chain reaction. After n cycles, there is an amplification factor of 2n. Typically PCR is run for 20-
30 cycles.
1 cycle 2 cycles 3 cycles 4 cycles 10 cycles 20 cycles
1 2 4 8 16 1024 1 048 576

molecule molecules molecules molecules molecules molecules molecules
PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified
millions of times with little effort. The product can be used for further studies, such as cloning,
electrophoresis, or gene probes. Because PCR can use such small samples it can be used in forensic
medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy
DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been
encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of
DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for
example in court cases.

2. Electrophoresis
This is a form of chromatography used to separate different pieces of DNA on the basis of their
length. It might typically be used to separate restriction fragments. The DNA samples are placed
into wells at one end of a thin slab of gel made of agarose or polyacrylamide, and covered in a
buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA
contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive
electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster
than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule,
the further down the gel it will move in a given time. At the end of the run the current is turned off.
place DNA
sample here Direction of movement
negative positive
electrode electrode
gel buffer
- well +
Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are two common
methods for doing this:
• The DNA can be stained with a coloured chemical such as azure A (which stains the DNA bands
blue), or a fluorescent molecule such as ethidium bromide (which emits coloured light when the
finished gel is illuminated with invisible ultraviolet light).
32
• The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as P,
then visualised using autoradiography. Ordinary photographic film is placed on top of the
finished gel in the dark for a few hours, and the radiation from any radioactive DNA on the gel
exposes the film. When the film is developed the position of the DNA shows up as dark bands on
the film. This method is extremely sensitive.
film after developing
In Dark Room
photographic film radiation
gel
invisible DNA spots large small

fragments fragments

3. DNA Sequencing
This means reading the base sequence of a length of DNA. DNA sequencing is based on a
beautifully elegant technique developed by Fred Sanger in Cambridge, and now called Sanger
Sequencing.
Radiolabelled DNA
1. Label 4 test tubes A, T, C and G. Into each test tube add: a DNA
Primer 4 polymerase
sample nucleotides
(TATGACCG)
sample of the DNA to be sequenced (containing many
millions of individual molecules) a radioactive primer (so
A T C G
the DNA can be visualised later on the gel), the four DNA
nucleotides and the enzyme DNA polymerase.
2. In each test tube add a small amount of a special modified

dideoxy nucleotide that cannot form a phosphodiester bond 1% A* 1% T* 1% C* 1% G*
and so stops further synthesis of DNA. Tube A has A T C G
dideoxy A (A*), tube T has dideoxy T (T*), tube C has

dideoxy C (C*) and tube G has dideoxy G (G*). The
dideoxy nucleotides are present at about 1% of the
concentration of the normal nucleotides.
3. Let the DNA polymerase synthesise many copies of the

DNA sample. From time to time at random a dideoxy
A T C G
nucleotide will be added to the growing chain and
synthesis of that chain will then stop. A range of DNA
molecules will be synthesised ranging from full length to
DNA fragments synthesised in each tube
very short. The important point is that in tube A, all the
TA* T* TATGAC* TATG*
fragments will stop at an A nucleotide. In tube T, all the TATGA* TAT* TATGACC* TATGACCG*
TATGACCG TATGACCG TATGACCG TATGACCG
fragments will stop at a T nucleotide , and so on.
4. The contents of the four tubes are now run side by side on A T C G
an electrophoresis gel, and the DNA bands are visualised

sequence
read
by autoradiography. Since the fragments are now sorted by in this
direction
length the sequence can simply be read off the gel starting
with the smallest fragment (just one nucleotide) at the
bottom and reading upwards.

There is now a modified version of the Sanger method called cycle sequencing, which can be
completely automated. The primers are not radiolabelled, but instead the four dideoxy nucleotides
are fluorescently labelled, each with a different colour (A* is green, T* is red, C* is blue and G* is
yellow). The polymerisation reaction is done in a single tube, using PCR-like cycles to speed up the
process. The resulting mixture is separated using capillary electrophoresis, which gives good
separation in a single narrow gel. The gel is read by a laser beam and the sequence of colours is
converted to a DNA sequence by computer program (like the screenshot below). This technique can
sequence an amazing 12 000 bases per minute.
Thousands of genes have been sequenced using these methods and the entire genomes of several
organisms have also been sequenced. A huge project to sequence the complete 3-billion base
sequence of the human genome was recently completed. This information is giving us
unprecedented knowledge about ourselves, and is likely to lead to dramatic medical and scientific
advances. Once a gene sequence is known the amino acid sequence of the protein that the DNA
codes for can also be determined, using the genetic code table. The sequence can also be compared
with DNA sequences from other individuals and even other species to work out relationships
between individuals or species.
These genome sequences represent vast amount of data that must be analysed and compared to
existing sequences. Powerful computers, huge databases and intelligent search programs are being
developed to deal with this data, which has led to a whole new branch of biology called
bioinformatics, and a new way of doing biology without touching a living thing: in silico.

4. Restriction Enzymes
These are enzymes that cut DNA at specific sites. They are properly called restriction
endonucleases because they cut phosphodiester bonds in the middle of the polynucleotide chain.
Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes
make a staggered cut in the two strands, forming sticky ends.
A A T T C A T G
G T A C
A C T G A A T T C A T G
sticky ends
T G A C T T A A G T A C
A C T G
T G A C T T A A
Restriction Enzyme
The cut ends are “sticky” because they have short stretches of single-stranded DNA with
complementary sequences. These sticky ends will stick (or anneal) to another sticky end by
complementary base pairing (i.e. with weak hydrogen bonds), but only if the sticky ends have both
been cut with the same restriction enzyme so that they have complementary sequences. Restriction
enzymes have highly specific active sites, and will only cut DNA at specific base sequences, 4-8
base pairs long, called recognition sequences.
Restriction enzymes are produced naturally by bacteria as a defence against viruses (they “restrict”
viral growth), but they are enormously useful in genetic engineering for cutting DNA at precise
places ("molecular scissors"). Short lengths of DNA cut out by restriction enzymes are called
restriction fragments. There are thousands of different restriction enzymes known, with over a
hundred different recognition sequences. Restriction enzymes are named after the bacteria species
they came from, so EcoR1 is from E. coli strain R, and HindIII is from Haemophilis influenzae.

5. DNA Ligase
This enzyme repairs broken DNA by joining two
A A T T C A T G
nucleotides in a DNA strand. Ligase is therefore a bit
G T A C
like DNA polymerase. It is commonly used in genetic sticky ends
A C T G
engineering to do the reverse of a restriction enzyme,
T G A C T T A A
i.e. to join together complementary restriction
fragments.
Complementary
base pairing
Two restriction fragments can anneal if they have

A C T G A A T T C A T G
complementary sticky ends, but only by weak
hydrogen bonds, which can quite easily be broken, say
by gentle heating. The backbone is still incomplete.
DNA Ligase
Ligase
DNA ligase completes the DNA backbone by forming
covalent phosphodiester bonds. Restriction enzymes A C T G A A T T C A T G
and DNA ligase can therefore be used together to join
lengths of DNA from different sources. Ligase
6. Plasmids
Plasmids are short circular bits of DNA found naturally in bacterial cells. In genetic engineering
plasmids are used as vectors. A vector is a length of DNA that carries the gene we want into a host
cell. A vector is needed because a length of DNA containing a gene on its own won’t actually do
anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated
when the cell divides, it won’t be expressed, and in fact it will probably be broken down pretty
quickly. A vector gets round these problems by having these properties:
• It is big enough to hold the gene we want (plus a few others), but not too big.
• It is circular (or more accurately a closed loop), so that it is less likely to be broken down
(particularly in prokaryotic cells where DNA is always circular).
• It contains control sequences, such as a replication origin and a transcription promoter, so that
the gene will be replicated, expressed, or incorporated into the cell’s normal genome.
• It contain marker genes, so that cells containing the vector can be identified.

Plasmids are the most common kind of vector, so we shall look at how they are used in some detail.
A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a
bacterial cell. Plasmids are copied separately from the main bacterial DNA when the cell divides, so
the plasmid genes are passed on to all daughter cells. They are also used naturally for exchange of
genes between bacterial cells (the nearest they get to sex), so bacterial cells will readily take up a
plasmid. Because they are so small, they are easy to handle in a test tube, and foreign genes can
quite easily be incorporated into them using restriction enzymes and DNA ligase.
The R plasmid
EcoRI One of the most common plasmids used is the R-plasmid. This
BamHI
plasmid contains a replication origin, several recognition sequences
PstI
ampicillin
resistance
for different restriction enzymes (with names like PstI and EcoRI),
gene SalI
tetracycline and two marker genes, which confer resistance to different
resistance
gene
antibiotics (ampicillin and tetracycline). The R plasmid gets its
replication
origin name from these resistance genes.
PvuII
The diagram below shows how DNA fragments can be incorporated into a plasmid using restriction
and ligase enzymes. The restriction enzyme used here (PstI) cuts the plasmid in the middle of one
of the marker genes (we’ll see why this is useful later). The foreign DNA anneals with the plasmid
and is joined covalently by DNA ligase to form a hybrid vector (in other words a mixture or hybrid
of bacterial and foreign DNA). Several other products are also formed: some plasmids will simply
re-anneal with themselves to re-form the original plasmid, and some DNA fragments will join
together to form chains or circles. These different products cannot easily be separated, but it doesn’t
matter, as the marker genes can be used later to identify the correct hybrid vector.
Restriction Enzyme original

hybrid plasmid
PstI plasmid
R-plasmid
DNA ligase +
Restriction Enzyme circularised

DNA
PstI
Foreign DNA

7. Transformation
Transformation means inserting new DNA (usually a plasmid) into a living cell (called a host cell),
which is thus genetically modified, or transformed. A transformed cell can replicate and express the
genes in the new DNA. DNA is a large molecule that does not readily cross cell membranes, so the
membranes must be made permeable in some way. There are different ways of doing this depending
on the type of host cell.
Transferring into cells in culture

• Heat Shock. Cells are incubated with the plasmid in a solution containing calcium ions at 0°C.
The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells
to take up the plasmid. This works well for bacterial and animal cells.
• Electroporation. Cells are subjected to a high-voltage pulse, which temporarily disrupts the
membrane and allows the plasmid to enter the cell. This is the most efficient method of
delivering genes to bacterial cells.
• Micro-Injection. A cell is held on a pipette under a microscope and the foreign DNA is injected
directly into the nucleus using an incredibly fine micro-pipette. This method is used where there
are only a very few cells available, such as fertilised animal egg cells. In the rare successful cases
the fertilised egg is implanted into the uterus of a surrogate mother and it will develop into a
normal animal, with the DNA incorporated into the chromosomes of every cell.
DNA
holding zygote injection

pipette pipette
Transferring into plant cells

• Gene Gun. This extraordinary technique fires microscopic gold particles coated with the foreign
DNA at the cells using a compressed air gun. It is designed to overcome the problem of the
strong cell wall in plant tissue, since the particles can penetrate the cell wall and the cell and
nuclear membranes, and deliver the DNA to the nucleus, where it is sometimes expressed.
• Plant Tumours. The plasmid is first inserted into a soil bacterium, and then plants are infected
with the bacterium. The bacterium inserts its plasmid into the plant cells' chromosomal DNA and
causes a "crown gall" tumour. These tumour cells can be cultured in the laboratory and whole

new plants grown from them by micropropagation. Every cell of these plants contains the foreign
gene.
Transferring into human cells in vivo

• Liposomes. Plasmids can be encased in liposomes, which are small membrane vesicles (see
module 1). The liposomes fuse with the cell membrane (and sometimes the nuclear membrane
too), delivering the DNA into the cell.
liposome
DNA fuses
part of cell membrane
• Viruses. The plasmid is first incorporated into a virus, which is then used to infect cells, carrying
the foreign gene along with its own genetic material. Since viruses rely on getting their DNA
into host cells for their survival they have evolved many successful methods, and so are an
obvious choice for gene delivery. The virus must first be genetically engineered to make it safe,
so that it can’t reproduce itself or make toxins. Two viruses are commonly used:
1. Adenoviruses are human viruses that causes respiratory diseases including the common cold.
Their genetic material is double-stranded DNA, and they are ideal for delivering genes to
living patients in gene therapy. Their DNA is not incorporated into the host’s chromosomes,
so it is not replicated, but their genes are expressed. The adenovirus is genetically altered so
that its coat proteins are not synthesised, so new virus particles cannot be assembled and the
host cell is not killed.
2. Retroviruses are a group of human viruses that include HIV. They are enclosed in a lipid
membrane and their genetic material is double-stranded RNA. On infection this RNA is
copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the
foreign genes are replicated into every daughter cell. After a certain time, the dormant DNA is
switched on, and the genes are expressed in all the host cells.

8. Genetic Markers
These are needed to identify cells that have successfully taken up a plasmid and so become
transformed. With most of the techniques above less than 1% of the cells actually take up the
plasmid, so a marker is needed to distinguish these cells from all the others. We’ll look at how to do
this with bacterial host cells, as that’s the most common technique.
A common marker, used in the R-plasmid, is a gene for resistance to an antibiotic such as
tetracycline. Bacterial cells taking up this plasmid can make this gene product and so are resistant to
this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal
untransformed cells, together with cells that have taken up DNA that’s not in a plasmid (99%) will
die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another
plate.
bacteria cells
(without plasmids)
most cells do untransformed cells are
not take up plasmid killed by tetracycline
spread thinly on
to agar plate
bacterial DNA containing
electroporation tetracycline
+
a few cells take
up plasmid and
become transformed transformed cells
plasmid vectors survive
9. Replica Plating
Replica plating is a simple technique for making an exact copy of an agar plate. A pad of sterile
cloth the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it.
Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar
plate, some cells will be deposited and colonies will grow in exactly the same positions on the new
plate. This technique has a number of uses, but the most common use in genetic engineering is to
help solve another problem in identifying transformed cells.
This problem is to distinguish those cells that have taken up a hybrid plasmid (with a foreign gene
in it) from those cells that have taken up the normal plasmid. This is where the second marker gene
(for resistance to ampicillin) is used. If the foreign gene is inserted into the middle of this marker
gene, the marker gene is disrupted and won't make its proper gene product. So cells with the hybrid

plasmid will be killed by ampicillin, while cells with the normal plasmid will be immune to
ampicillin. Since this method of identification involves killing the cells we want, we must first
make a master agar plate and then make a replica plate of this to test for ampicillin resistance.
replica (ampicillin) plate

master (tetracycline) plate
press to press to
pick up cells deposit cells
cells stuck to
sterile velvet surface
identify these missing colonies

colonies indicate cells
with hybrid vector
grid pattern of colonies transfer to
makes identification easier another plate
Once the colonies of cells containing the correct hybrid plasmid have been identified, the
appropriate colonies on the master plate can be selected and grown on another plate.
The R-plasmid with its antibiotic-resistance genes dates from the early days of genetic engineering
in the 1970s. Scientists are now worried about pathogenic bacteria gaining antibiotic resistance, so
have stopped using this technique. In recent years better plasmids with different marker genes have
been developed that do not kill the desired cells, and so do not need a replica plate. These new
marker genes make an enzyme (actually lactase) that converts a colourless substrate in the agar
medium into a blue-coloured product that can easily be seen. So cells with a normal plasmid turn
blue on the correct medium, while those with the hybrid plasmid can't make the enzyme and stay
white. These white colonies can easily be identified and transferred to another plate. Another
marker gene, transferred from jellyfish, makes a green fluorescent protein (GFP).

10. Fermenters
Once colonies of transformed bacteria have been identified on an agar plate they can at last be used
to make the genetically-engineered product. To do this commercially the bacteria need to be grown
in very large quantities in fermenters. Fermenters are so-called because they were developed from
the large vessels used in breweries where yeast ferments sugar to alcohol. First bacteria cells are
transferred from the agar plate to a liquid medium in a culture flask, where the cells grow for a few
days, then the culture flask is used to inoculate a larger laboratory fermenter, where the bacteria
grow further. Finally the culture is used to inoculate a huge industrial fermenter. This process is
called scaling up:
agar plate culture flask laboratory fermenter industrial fermenter

(0.5 L) (10 L) (10000 L)
All these steps must be done under sterile conditions to ensure that no other microbes contaminate
the cultures. In addition the conditions in the fermenters must be very strictly controlled to
maximise the growth rate of the bacteria.
• Oxygen for respiration is provided by bubbling air through the fermenter and rapid stirring.
• Temperature is controlled using a thermostated water jacket.
• Pressure increase due the release of gases is controlled using a vent.
• pH is controlled by automatically adding acid or alkali.
• Sugars and other materials can be added as required by the bacteria.
In industrial fermenters these conditions are constantly monitored using probes and controlled by
computers. Under these optimal conditions the bacteria can grow very quickly. After a few days
growth the culture is run off from the fermentation vessel, and the product is purified from the
mixture by a process called downstream processing.

Applications of Genetic Engineering

We have now looked at some of the many techniques used by genetic engineers. What can be done
with these techniques? By far the most numerous applications are still as research tools, and the
techniques above are helping geneticists to understand complex genetic systems. Despite all the
hype, genetic engineering still has very few successful commercial applications, although these are
increasing each year. The applications so far can usefully be considered in three groups.
• Gene Products using genetically modified organisms (usually microbes) to produce

chemicals (usually proteins) for medical or industrial applications.
• New Phenotypes using gene technology to alter the characteristics of organisms (usually farm
animals or crops)
• Gene Therapy using gene technology on humans to treat a disease
Gene Products
The biggest and most successful kind of genetic engineering is the production of gene products.
These products are of medical, agricultural or commercial value. This table shows a few of the
examples of genetically engineered products that are already available.
Product Use Host Organism

Insulin human hormone used to treat diabetes bacteria /yeast
HGH human growth hormone, used to treat dwarfism bacteria
Enkephalin human hormone plants
BST bovine growth hormone, used to increase milk yield of cows bacteria
Factor VIII human blood clotting factor, used to treat haemophiliacs bacteria
Anti-thrombin anti-blood clotting agent used in surgery goats
Penicillin antibiotic, used to kill bacteria fungi / bacteria
Vaccines hepatitis B antigen, for vaccination yeast
Antibodies research and clinical use goats / plants
AAT enzyme inhibitor used to treat cystic fibrosis and emphysema sheep
α-glucosidase enzyme used to treat Pompe’s disease rabbits
DNAse enzyme used to treat CF bacteria
rennin enzyme used in manufacture of cheese bacteria /yeast
cellulase enzyme used in paper production bacteria
PHB biodegradable plastic plants
The products are mostly proteins, which are produced directly when a gene is expressed, but they
can also be non-protein products produced by genetically-engineered enzymes. The basic idea is to
transfer a gene (often human) to another host organism (usually a microbe) so that it will make the

gene product quickly, cheaply and ethically. It is also possible to make “designer proteins” by
altering gene sequences, but while this is a useful research tool, there are no commercial
applications yet.
Since the end-product is just a chemical, in principle any kind of organism could be used to produce
it. By far the most common group of host organisms used to make gene products are the bacteria,
since they can be grown quickly and the product can be purified from their cells. Unfortunately
bacteria cannot not always make human proteins, and recently animals and even plants have also
been used to make gene products. In neither case is it appropriate to extract the product from their
cells, but in animals the product can be secreted in milk or urine, while in plants the product can be
secreted from the roots. This table shows some of the advantages and disadvantages of using
different organisms for the production of genetically-engineered gene products.
Type of
Advantages Disadvantages
organism
Prokaryotes no nucleus so DNA easy to modify; have can’t splice introns; no post-
(Bacteria) plasmids; small genome; genetics well translational modification; small
understood; asexual so can be cloned; small gene size
and fast growing; easy to grow
commercially in fermenters; will use cheap
carbohydrate; few ethical problems.
Eukaryotes can splice introns; can do post-translational Do not have plasmids (except yeast);
modifications; can accept large genes often diploid so two copies of genes
may need to be inserted; control of
expression not well understood.
Fungi (yeast, asexual so can be cloned; haploid, so only can’t always make animals’ gene
mould) one copy needed; can be grown in vats products
Plants photosynthetic so don’t need much feeding; cell walls difficult to penetrate by
can be cloned from single cells; products vector; slow growing; multicellular
can be secreted from roots or in sap.
Animals most likely to be able to make human multicellular; slow growing;

(pharming) proteins; products can be secreted in milk or expensive to produce
urine
We’ll look at some examples in detail.

Human Insulin
Insulin is a small protein hormone produced by the pancreas to regulate the blood sugar
concentration. In the disease insulin-dependent diabetes the pancreas cells don’t produce enough
insulin, causing wasting symptoms and eventually death. The disease can be successfully treated by
injection of insulin extracted from the pancreases of slaughtered cows and pigs. However the
insulin from these species has a slightly different amino acid sequence from human insulin and this
can lead to immune rejection and side effects.
The human insulin gene was isolated, cloned and sequenced in the 1970s, and so it became possible
to insert this gene into bacteria, who could then produce human insulin in large amounts.
Unfortunately it wasn’t that simple. In humans, pancreatic cells first make pro-insulin, which then
undergoes post-translational modification to make the final, functional insulin. Furthermore the
human insulin gene contain two introns.
e protein
i post-translational S
transcription e remove introns translation S modification
S
S
i
e
insulin gene primary transcript mature mRNA pro-insulin insulin
mRNA
Bacterial cells cannot do post-translational modification, nor can they splice out introns. Eventually
a synthetic cDNA gene (with no introns) was made and inserted into the bacterium E. coli, which
made pro-insulin, and the post-translational conversion to insulin was carried out chemically.
expression chemical S
reverse transcriptase in bacteria S modification
S
S
insulin mRNA cDNA pro-insulin insulin

(no introns) (no introns)
This technique was developed by Eli Lilly and Company in 1982 and the product, “humulin”
became the first genetically-engineered product approved for medical use.
In the 1990s the procedure was improved by using the yeast Saccharomyces cerevisiae instead of E.
coli. Yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the
production of human insulin. However another company has developed a method of converting pig

insulin into human insulin by chemically changing a few amino acids, and this turns out to be
cheaper than the genetic engineering methods.
Human Growth Hormone (HGH)

HGH is a protein hormone secreted by the pituitary gland, which stimulates tissue growth. Low
production of HGH in childhood results in pituitary dwarfism. This can be treated with HGH
extracted from dead humans, but as the treatment caused some side effects, such as Creutzfeldt-
Jacod disease (CJD), the treatment was withdrawn. The HGH gene has been cloned and an artificial
cDNA gene has been inserted into E. coli. A signal sequence has been added which not only causes
the gene to be translated but also causes the protein to be secreted from the cell, which makes
purification much easier. This genetically engineered HGH is produced by Genentech and can
successfully restore normal height to children with HGH deficiency.
Bovine Somatotrophin (BST)

BST is a growth hormone produced by cattle. The gene has been cloned in bacteria by the company
Monsanto, who now produce large quantities of BST. In the USA cattle are often injected with BST
every 2 weeks, resulting in a 10% increase in mass in beef cattle and a 25% increase in milk
production in dairy cows. BST was tested in the UK in 1985, but it was not approved and its use is
currently banned in the EU. This is partly due to public concerns and partly because there is already
overproduction of milk and beef in the EU, so greater production is not necessary.
Antibiotics
The first antibiotic, penicillin, was discovered in the fungus Penicillium, but most antibiotics now
are produced by bacteria, particularly the genus Streptomyces, which produce streptomycin,
tetracycline and erythromycin. Attempts are being made to engineer Streptomyces bacteria to
produce better antibiotics, or to produce them in larger quantities. A hybrid antibiotic has been
made by combining genes from different strains of Streptomyces, but this has proved to be no better
than existing antibiotics. So far genetic engineering has only been used successfully to increase the
production of existing antibiotics by overexpression of the genes involved.

Rennin
Rennin (also known as chymosin) is an enzyme used in the production of cheese. It is produced in
the stomach of juvenile mammals (including humans) and it helps the digestion of the milk protein
caesin by solidifying it so that is remains longer in the stomach. Traditionally the cheese industry
has used rennin obtained from the stomach of young calves when they are slaughtered for veal, but
there are moral and practical objections to this source. Now an artificial cDNA gene for rennin has
been made from mRNA extracted from calf stomach cells, and this gene has been inserted into a
variety of microbes such as the bacterium E. coli and the fungus Aspergillus niger. The rennin
extracted from these microbes has been very successful and 90% of all hard cheeses in the UK are
made using microbial rennin. Sometimes (though not always) these products are labelled as
“vegetarian cheese”.
α-1-antitrypsin)
AAT (α
AAT is a human protein made in the liver and found in the blood. As the name suggests it is an
inhibitor of protease enzymes like trypsin and elastase. There is a rare mutation of the AAT gene (a
single base substitution) that causes AAT to be inactive, and so the protease enzymes to be
uninhibited. The most noticeable effect of this is in the lungs, where elastase digests the elastic
tissue of the alveoli, leading to the lung disease emphysema. This condition can be treated by
inhaling an aerosol spray containing AAT so that it reaches the alveoli and inhibits the elastase
there.
AAT for this treatment can be extracted from blood donations, but only in very small amounts. The
gene for AAT has been found and cloned, but AAT cannot be produced in bacteria because AAT is
a glycoprotein, which means that it needs to have sugars added by post translational modification.
This kind of modification can only be carried out by animals, and AAT is now produced by
genetically-modified sheep. In order to make the AAT easy to extract, the gene was coupled to a
promoter for the milk protein β-lactoglubulin. Since this promoter is only activated in mammary
gland cells, the AAT gene will only be expressed in mammary gland cells, and so will be secreted
into the sheep's milk. This makes it very easy to harvest and purify without harming the sheep. The
first transgenic sheep to produce AAT was called Tracy, and she was produced by PPL
Pharmaceuticals in Edinburgh in 1993. This is how Tracy was made:

1. A female sheep is given a fertility drug to stimulate her egg production,

and several mature eggs are collected from her ovaries.
2. The eggs are fertilised in vitro.
3. A plasmid is prepared containing the gene for human AAT and the
promoter sequence for β-lactoglobulin. Hundreds of copies of this
plasmid are microinjected into the nucleus of the fertilised zygotes (see
p44). Only a few of the zygotes will be transformed, but at this stage you
can’t tell which.
4. The zygotes divide in vitro until the embryos are at the 16-cell stage.
5. The 16-cell embryos are implanted into the uterus of surrogate mother
ewes. Only a few implantations result in a successful pregnancy.
6. Test all the offspring from the surrogate mothers for AAT production in
their milk. This is the only way to find if the zygote took up the AAT
gene so that it can be expressed. About 1 in 20 eggs are successful.
7. Collect milk from the transgenic sheep for the rest of their lives. Their
milk contains about 35 g of AAT per litre of milk.
8. Purify the AAT, which is worth about £50 000 per mg.
AAT
9. Breed from the transgenic sheep in order to build up a herd of them.

New Phenotypes
This means altering the characteristics of organisms by genetic engineering. The organisms are
generally commercially-important crops or farm animals, and the object is to improve their quality
in some way. This can be seen as a high-tech version of selective breeding, which has been used by
humans to alter and improve their crops and animals for at least 10 000 years. Nevertheless GMOs
have turned out to be a highly controversial development. None of these new phenotypes is on the
specification, but these are some of the most active uses of genetic engineering and are often in the
news, so this table gives an idea of what is being done.
organism Modification
long life tomatoes There are two well-known projects, both affecting the gene for the enzyme
polygalactourinase (PG), a pectinase that softens fruits as they ripen. Tomatoes
that make less PG ripen more slowly and retain more flavour. The American
“Flavr Savr” tomato used antisense technology to silence the gene, while the
British Zeneca tomato disrupted the gene. Both were successful and were on
sale for a few years, but neither is produced any more.
Insect-resistant Genes for various powerful protein toxins have been transferred from the
crops bacterium Bacillus thuringiensis to crop plants including maize, rice and
potatoes. These Bt toxins are thousands of times more powerful than chemical
insecticides, and since they are built-in to the crops, insecticide spraying (which
is non-specific and damages the environment) is unnecessary.
virus-resistant Gene for virus coat protein has been cloned and inserted into tobacco, potato
crops and tomato plants. The coat protein seems to “immunise” the plants, which are
much more resistant to viral attack.
herbicide The gene for resistance to the herbicide BASTA has been transferred from
resistant crops Streptomyces bacteria to tomato, potato, corn, and wheat plants, making them
resistant to BASTA. Fields can safely be sprayed with this herbicide, which
will kill all weeds, but not the crops. However, this means continued use of
agrochemicals, so is controversial.
pest-resistant The gene for an enzyme that synthesises a chemical toxic to weevils has been
legumes transferred from Bacillus bacteria to The Rhizobium bacteria that live in the
root nodules of legume plants. These root nodules are now resistant to attack by
weevils.
Nitrogen-fixing This is a huge project, which aims to transfer the 15-or-so genes required for
crops nitrogen fixation from the nitrogen-fixing bacteria Rhizobium into cereals and
other crop plants. These crops would then be able to fix their own atmospheric
nitrogen and would not need any fertiliser. However, the process is extremely
complex, and the project is nowhere near success.

crop Proteins in some crop plants, including wheat, are often deficient in essential
improvement amino acids (which is why vegetarians have to watch their diet so carefully), so
the protein genes are being altered to improve their composition for human
consumption.
mastitis-resistant The gene for the enzyme lactoferrin, which helps to resists the infection that
cattle causes the udder disease mastitis, has been introduced to Herman – the first
transgenic bull. Herman’s offspring inherit this gene, do not get mastitis and so
produce more milk.
tick-resistant The gene for the enzyme chitinase, which kills ticks by digesting their
sheep exoskeletons, has bee transferred from plants to sheep. These sheep should be
immune to tick parasites, and may not need sheep dip.
Fast-growing The human growth hormone gene has been transferred to sheep, so that they
sheep produce human growth hormone and grow more quickly. However they are
more prone to infection and the females are infertile.
Fast-growing fish A number of fish species, including salmon, trout and carp, have been given a
gene from another fish (the ocean pout) which activates the fish’s own growth
hormone gene so that they grow larger and more quickly. Salmon grow to 30
times their normal mass at 10 time the normal rate.
environment Genes for enzymes that digest many different hydrocarbons found in crude oil
cleaning have been transferred to Pseudomonas bacteria so that they can clean up oil
microbes spills.

Gene Therapy
Gene therapy is perhaps the most significant, and most controversial kind of genetic engineering. It
is also the least well-developed. The idea of gene therapy is to genetically alter humans in order to
treat a disease. This could represent the first opportunity to cure incurable diseases. Note that this is
quite different from using genetically-engineered microbes to produce a drug, vaccine or hormone
to treat a disease by conventional means. Gene therapy means altering the genotype of a tissue or
even a whole human.
Cystic Fibrosis
Cystic fibrosis (CF) is the most common genetic disease in the UK, affecting about 1 in 2500. It is
caused by a mutation in the gene for protein called CFTR (Cystic Fibrosis Transmembrane
Regulator). The gene is located on chromosome 7, and the usual mutation is a deletion of three
bases, removing one amino acid out of 1480 amino acids in the protein. CFTR is a chloride ion
channel protein found in the cell membrane of epithelial tissue cells, which line all the open spaces
in the human body (such as lungs and gut). The mutation stops the protein channel from working,
so chloride ions cannot cross the cell membrane.
Normal Bronchiole Cystic Fibrosis Bronchiole
Cl- Cl-
mutated
CFTR CFTR
H2O less H2O
high Ψ low Ψ
alveolus
decreased Ψ increased Ψ
runny thick sticky
mucus mucus
epithelial cell Alveolar epithelial cell Alveolar
air space air space
Chloride ions build up inside these epithelial cells, decreasing their water potential (Ψ). Less water
therefore diffuses out of these cells to the mucus outside, leaving the mucus drier and more sticky
than normal. This sticky mucus block the tubes into which it is secreted, such as the small intestine,
pancreatic duct, bile duct, sperm duct, sweat ducts, bronchioles and alveoli.
These blockages lead to the symptoms of CF: breathlessness (since the bronchioles are narrowed);
lung infections such as bronchitis and pneumonia (since the cilia can't move the thick mucus and so
trapped bacteria aren't killed); poor digestion (since the bile and pancreatic ducts are blocked); poor
absorption (since thick mucus slows diffusion in the ileum) and infertility (since the sperm ducts are

blocked). Of these symptoms the lung effects are the most serious, causing 95% of deaths. CF is
always fatal, though life expectancy has increased from 1 year to about 20 years due to modern
treatments. These treatments include physiotherapy many times each day to dislodge mucus from
the lungs; antibiotics to fight infections; DNAse and protease drugs to loosen the mucus; enzymes
to help food digestion and even a heart-lung transplant.
Given these complicated (and ultimately unsuccessful) treatments, CF is a good candidate for gene
therapy, and was one of the first diseases to be tackled this way. The gene for CFTR was identified
and cloned in 1989. The idea is to deliver copies of this good gene to the epithelial cells of the lung,
where they can be incorporated into the nuclear DNA and make functional CFTR chloride channels.
If about 10% of the epithelial cells could be genetically modified, this would allow enough chloride
ions to be transported to relieve the symptoms of the disease. Note that gene therapy doesn't alter or
replace the existing mutated gene, which will still continue to make useless CFTR channels. But in
addition, the new gene will make working CFTR channels, which will allow the epithelial cells to
function normally.
Two methods of delivery are being tried: liposomes and adenoviruses (see p 45), both delivered
with an aerosol inhaler, like those used by asthmatics. Clinical trials are currently underway, but as
yet no therapy has been shown to be successful.
SCID
Severe Combined immunodeficiency Disease (SCID) is a rare genetic disease that affects the
immune system. It is caused by a mutation in the gene for the enzyme adenosine deaminase (ADA).
Without this enzyme white blood cells cannot be made, so sufferers have almost no effective
immune system and would quickly contract a fatal infection unless they spend their lives in sterile
isolation (SCID is also known as “baby in a bubble syndrome”). Gene therapy has been attempted
with a few children in the USA and UK by surgically removing bone marrow cells (which
manufacture white blood cells in the body) from the patient, transfecting them with a genetically-
engineered virus containing the ADA gene, and then returning the transformed cells to the patient.
The hope is that these transformed cells will multiply in the bone marrow and make white blood
cells. The trials are still underway, so the success is unknown.

The Future of Gene Therapy

Gene therapy is in its infancy, and is still very much an area of research rather than application. No
one has yet been cured by gene therapy, but the potential remains enticing. Gene therapy need not
even be limited to treating genetic diseases, but could eventually also help in treating infections and
environmental diseases e.g.:
• White blood cells have been genetically modified to produce tumour necrosis factor (TNF), a
protein that kills cancer cells, making these cells more effective against tumours.
• Genes could be targeted directly at cancer cells, causing them to die, or to revert to normal cell
division.
• White blood cells could be given antisense genes for HIV proteins, so that if the virus infected
these cells it couldn’t reproduce.
It is important to appreciate the different between somatic cell therapy and germ-line therapy.
• Somatic cell therapy means genetically altering specific body (or somatic) cells, such as trachea
epithelial cells, bone marrow cells, pancreas cells, or whatever, in order to treat the disease. This
therapy may treat the disease in the patient, but any genetic changes will not be passed on the
offspring of the patient.
• Germ-line therapy means genetically altering those cells (sperm cells, sperm precursor cells, ova,
ova precursor cells, zygotes or early embryos) that will pass their genes down the “germ-line” to
future generations. Alterations to any of these cells will affect every cell in the resulting human,
and in all his or her descendants.
Germ-line therapy would be highly effective, but is also potentially dangerous (since the long-term
effects of genetic alterations are not known), unethical (since it could easily lead to eugenics) and
immoral (since it could involve altering and destroying human embryos). It is currently illegal in the
UK and most other countries, and current research is focussing on somatic cell therapy only. All
gene therapy trials in the UK must be approved by the Gene Therapy Advisory Committee (GTAC),
a government body that reviews the medical and ethical grounds for a trial. Germ-line modification
is allowed with animals, and indeed is the basis for producing GMOs.

Module 2 Notes

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Module 2 - Genetics - page 1

These notes may be used freely by A level biology students

Neil C Millar (nmillar@ntlworld.com)

HGS A-level notes NCM/6/07

HGS A-level notes NCM/6/07

HGS A-level notes NCM/6/07

HGS A-level notes NCM/6/07

that the nucleotides can join together in any order along

A polynucleotide has a free phosphate group at one 3' 2'

HGS A-level notes NCM/6/07

HGS A-level notes NCM/6/07

HGS A-level notes NCM/6/07

Replication - DNA Synthesis

HGS A-level notes NCM/6/07

The Meselson-Stahl Experiment

1. Grow bacteria light DNA

This is the crucial step. The DNA

14 light DNA After two generations the DNA is

HGS A-level notes NCM/6/07

• RNA is made of ribose nucleotides instead of deoxyribose nucleotides

Messenger RNA (mRNA)

Ribosomal RNA (rRNA)

Transfer RNA (tRNA)

HGS A-level notes NCM/6/07

Transcription - RNA Synthesis

rough endoplasmic reticulum ribosomes

HGS A-level notes NCM/6/07

Introns and Exons

exon 1 exon 2 exon 3 exon 4 exon 5

HGS A-level notes NCM/6/07

The Genetic Code

The Genetic Code (mRNA codons)

HGS A-level notes NCM/6/07

Translation - Protein Synthesis

3. The next amino acid-tRNA attaches to the met leu

adjacent mRNA codon (CUG, leu in this case) by

new amino acid-tRNA can attach. The free tRNA

6. The polypeptide chain elongates one amino acid

the finished protein.

HGS A-level notes NCM/6/07

Regulation of Gene Expression

HGS A-level notes NCM/6/07

Compound A enzyme P Compound B enzyme Q Compound C

HGS A-level notes NCM/6/07

So there are three possible phenotypic effects of a mutation:

There are three kinds of gene mutation, shown in this diagram:

mRNA . . . C G C G U U U C C . . . (part of original gene)

SUBSTITUTION DELETION INSERTION

HGS A-level notes NCM/6/07

Mutation Rates and Mutagens

HGS A-level notes NCM/6/07

DNA and Chromosomes

histone proteins centromere

DNA double helix

HGS A-level notes NCM/6/07

Karyotypes and Homologous Chromosomes

This display is called a karyotype, and it shows several features:

HGS A-level notes NCM/6/07

Sometimes the chromosomes in a cell nucleus are represented by

because the chromatin is usually uncoiled. Each ideogram

The only time chromosomes can actually be seen is during cell

two identical chromatids, because each DNA molecule has now

Chromatin DNA + histone complex during interphase

dideoxy A (A), tube T has dideoxy T (T), tube C has