[Link].

uk

3.8.1 Alteration of the sequence of bases in DNA can alter the

structure of proteins
Gene mutations

• Change in base sequence of DNA

• Can arise spontaneously during DNA replication, during interphase of the cell cycle

Mutagenic agents

• Increase the rate of gene mutation (above the rate of naturally occurring mutations)
• Examples:
• Ionising radiation (gamma and X-rays)
• Carcinogens e.g. mustard gas
• Some viruses

Types of gene mutations and their effects on amino acid sequences

Description Effect on amino acid sequence in the encoded

polypeptide
Substitution 1 base replaced • No change due to degenerate nature of genetic
with another code (see below table)
OR
• 1 triplet / codon change → 1 amino acid change
Addition 1 or more bases • Frameshift; triplets / codons change
added to base downstream of mutation → amino acid
sequence sequence changes (see below table)
OR
• If multiple of 3 bases added – no frameshift, but
extra triplets / codons → extra amino acids
Deletion 1 or more bases • Frameshift; triplets / codons change
lost from base downstream of mutation → amino acid
sequence sequence changes (see below table)
OR
• If multiple of 3 bases lost – no frameshift, but
missing triplets / codons → missing amino acids
Inversion A sequence of • No frameshift because number of bases stays
bases is separated the same
from DNA and • Triplets / codons in inverted region change →
inserted at the sequence of amino acids encoded by inverted
same position, region change
backwards

(table continued on next page)

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Duplication A sequence of • Frameshift; triplets / codons change

bases is inserted downstream of mutation → amino acid
twice, or multiple sequence changes (see below table)
times OR
• If multiple of 3 bases added – no frameshift, but
extra triplets / codons → extra amino acids
Translocation Sequence of bases • Significant impact on gene expression and
taken out and amino acid sequences at original and new
inserted at a location
different position
on the same, or a
different
chromosome

Effects of mutations: not all affect the

Figure 8.1 The genetic code (mRNA). If the
order of amino acids in the polypeptide
codon GUU changes to GUC, GUA or GUG, it
will still code for the amino acid valine.
• Some gene mutations (substitution)
change only 1 codon
• New codon might still code for same
amino acid because genetic code is
degenerate (meaning the same
amino acid can be coded for by more
than one triplet), as shown in fig. 8.1.
• Also, some gene mutations occur in
the introns (non-coding sequences
within genes) and therefore won’t
affect amino acid sequences

Effects of mutations: frame shift

• A frameshift occurs when gene

Figure 8.2 Frameshift.
mutations such as insertion or deletion
change the number of nucleotides by
any number not divisible by 3
• This shifts the way the genetic code is
read, so all the DNA triplets / mRNA
codons downstream from the
mutation change
• The sequence of amino acids encoded
changes accordingly and the effects on
the encoded polypeptide are
significant (shown in fig. 8.2)

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Stop codons

• There are 3 stop codons in the genetic code (UAA, UGA, UAG)
• Unlike other codons, these don’t code for amino acids, so they terminate translation
• A mutation (substitution or frameshift) may create a premature stop codon and result in
the production of a shorter and often non-functional polypeptide

How mutations can lead to the production of a non-functional protein / enzyme

1. Change in base / triplet sequence of DNA / gene

2. Changes sequence of codons on mRNA
3. Changes sequence of amino acids in primary structure of polypeptide
4. Changes position of hydrogen / ionic / disulphide bonds in protein tertiary structure
5. Changes tertiary structure / shape of protein and in the case of enzymes, the active site
will change shape
6. In the case of enzymes, the substrate will be unable to bind to active site and form an
enzyme-substrate complex

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3.8.2 Gene expression is controlled by a number of features

[Link] Most of a cell’s DNA is not translated

Stem cells

Unspecialised cells capable of:

• Self-renewal; can divide to replace themselves
• Specialisation/differentiation; can develop into other types of cell

Stem cell specialisation

1. Stimulus e.g. chemical

2. Causes selective activation of genes – some genes activated while others inactivated
• E.g. muscle cells genes coding for actin and myosin need to be activated
3. mRNA only transcribed from active genes → translated on ribosomes = proteins
4. These proteins modify cell permanently and determine cell structure / function

Potency – types of stem cells

• Totipotent cells
• Occur for a limited time in early mammalian embryos
• Can divide and differentiate into every cell type in body (including the cells that
support the embryo, such as the placenta)
• Pluripotent cells
• Found in embryos
• Can divide and differentiate into most cell types (every cell type in body but not
the cells of the placenta)
• Multipotent cells
• Found in mature mammals
• Can divide and differentiate into a limited number of cell types
• E.g. multipotent cells in bone marrow can differentiate into different types of
blood cell
• Unipotent cells
• Found in mature mammals
• Can divide and differentiate into just one cell type
• Example: cardiomyocytes (cardiac muscle cells) can be made from unipotent
stem cells

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Stem cells in medicine

• Regrow damaged tissues in accidents (i.e. skin grafts) or by disease (i.e. neuro-
degenerative diseases, Parkinson’s disease)
• Lots of potential links to topic 6 here e.g. B cells of the pancreas in type 1 diabetes
• Drug testing – used to grow artificial tissues
• Developmental biology research – provide insight into embryological development

Induced pluripotent stem cells

• How they are produced

1. Produced from adult somatic cells (non-pluripotent cells or fibroblasts)
2. Specific protein transcription factors associated with pluripotency put into cells,
causing the cell to express genes associated with pluripotency (reprogrammed)
3. Cells cultured
4. = Induced pluripotent stem cells
• Used in medical treatment instead of embryonic cells…
✓ No immune rejection as can be made using patient’s own cells
✓ Overcome some ethical issues with using embryonic stem cells e.g. no
destruction of embryo and adult can give permission

Evaluate use of stem cells in treating human disorders

Evaluate – look at both sides; scientific and ethical problems

For:
• Use of embryonic stem cells
• Tiny ball of cells, incapable of feeling pain, not equivalent to a human
• Would otherwise be destroyed (if from infertility treatment which creates more
than needed)
• Duty to apply knowledge to relieve human suffering

Against:
• Use of embryonic stem cells
• Embryo is a potential human; should be given rights
• Scientific
• Induced pluripotent stem cells – cannot yet reliably reprogramme stem cells
• Could begin to multiply out of control, and cause tumours

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[Link] Regulation of transcription and translation

Gene expression: transcription and translation – recap 3.4.2 DNA and protein synthesis

Regulation of transcription: transcription factors

• Transcription factors are proteins

• Move from cytoplasm → nucleus
• Bind to DNA at a specific DNA base
sequence on a promotor region
(near start / upstream of target
gene)
• Stimulate (‘activator’) or inhibit
(‘repressor’) transcription (the
production of mRNA) of target
gene(s) (by helping or preventing
RNA polymerase binding)

The role of oestrogen in initiating transcription

1. Oestrogen, a steroid hormone, can diffuse across the phospholipid bilayer of the cell-
surface membrane as it’s lipid soluble.
2. In cytoplasm, oestrogen binds to a
receptor of an inactive
transcription factor, forming a
hormone-receptor complex
3. Inactive transcription factor
changes shape, resulting in active
transcription factor
4. Diffuses from cytoplasm into
nucleus and binds to specific DNA
base sequence on a promotor
region
5. Stimulates transcription of genes
by helping RNA polymerase to bind

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Regulation at the chromosomal level: epigenetics

Nucleosome: DNA wrapped around histone proteins

• How closely the DNA and histone are packed together affects transcription

Epigenetics: heritable changes in gene function (expression) without changes to the base
sequence of DNA, caused by changes in the environment
• Epigenetic changes (‘chemical tags’) can inhibit transcription:
• Methylation of DNA
• Methyl groups added to cytosine bases in DNA
• Nucleosomes pack more tightly together → prevents transcription
factors binding; genes not transcribed (RNA polymerase can’t bind)
• Irreversible
• Decreased acetylation of associated histones
• Decreased acetylation of increases positive charge of histones
• Histones bind DNA (which is negatively charged) more tightly →
preventing transcription factors binding; genes not transcribed
• Reversible

Relevance of epigenetics on disease development and treatment, especially cancer

• Epigenetic changes that increase the expression of an oncogene, or that silence a

tumour suppressor gene, can lead to tumour development (see next section)
• Tests can be used to see if a patient has abnormal levels of methyl and acetyl – early
indicator of cancer (called a biomarker)
• Could be manipulated to treat cancer i.e. drugs to prevent histone acetylation / DNA
methylation that may have caused these genes to be switched on/off, resulting in cancer

Regulation of translation: RNA interference (RNAi)

RNA interference (RNAi): RNA molecules inhibit translation of mRNA produced by

transcription (gene is ‘switched on’ but encoded protein not produced = ‘silenced’ gene)

• RNAi can be moderated by either siRNA or miRNA (subtle differences between the two)
1. Micro-RNA (miRNA)
• Formed as hair-pin bends of RNA but processed into single-strands 22-26
nucleotides long, both become incorporated into a protein-based RISC (RNA-
induced silencing complex)
2. Small interfering RNA (siRNA)
• Formed as double-stranded molecules 21-25 bp long, one strand incorporated
into a protein-based RISC
• Single-stranded miRNA/siRNA within a RISC binds to a molecule of mRNA containing a
sequence of bases complementary to its own → mRNA hydrolysed / translation stopped
• miRNA expression deregulated in many human diseases including cancer → offer
opportunities as biomarkers and novel therapies

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[Link] Gene expression and cancer

Cancer

• Uncontrolled cell division → tumour

• Not all tumours are cancerous, they can be classified as:
o Benign (non-cancerous; don’t spread) or
o Malignant (cancerous; spread easily throughout the body via metastasis)

Main characteristics of benign and malignant tumours

Benign tumours Malignant

Grow slowly (rare mitoses) Grow rapidly (more frequent and / or
abnormal mitoses)
Well differentiated / specialised (cells retain Cells become unspecialised / poorly
function and normal shape, regular nuclei) differentiated
Normal, regular nuclei Irregular, larger/darker nuclei
Well defined borders/boundary; cell Irregular / poorly defined borders and not
adhesion molecules stick cells together and encapsulated; cells break off (+ grows
to a particular tissue, often surrounded by a projections into surrounding tissues) so
capsule so remain within tissues metastasis occurs
Easy to treat - can normally be removed by Removed by radiotherapy / chemotherapy as
surgery, rarely returns well as surgery; can be life threatening and
recurrence more likely

The role of tumour suppressor genes and oncogenes in the development of tumours,
including their abnormal methylation

Tumour suppressor genes (common example – p53)

• Normal function
• Code for proteins involved in control of cell division
• In particular, stopping cell cycle (when DNA damage detected)
• Also involved in causing self-destruction of cell (apoptosis) (where damaged DNA
cannot be repaired)
• Role in development of tumours
• Mutation alters amino acid sequence and tertiary structure of protein = non-
functional protein
• Or increased methylation prevents transcription / expression of protein
• Damaged DNA not repaired / cells not killed; uncontrolled cell division
• Note – would need 2 mutated alleles

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(Proto)- oncogenes
• Normal function
• Code for proteins involved in control of cell division
• In particular, stimulating cell division (when growth factors attach to receptors
on cell membrane, so cell division is required)
• Role in development of tumours
• Mutation could turn it into permanently activated oncogene
• Decreased methylation / increased acetylation causes excess transcription
• Cell division permanently activated; rapid / uncontrolled cell division
• Note – only need 1 mutated allele

The role of increased oestrogen concentrations in the development of some breast

cancers

• Areas of high oestrogen conc. such as adipose tissues in breasts, cell division
uncontrolled
• Growth of cancer minimised with drugs blocking production / action of oestrogens in the
breasts e.g. Tamoxifen prevents oestrogen binding to receptor

You should be able to evaluate evidence showing correlations between genetic and
environmental factors and various forms of cancer

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3.8.3 Using genome projects

Genome: the complete set of genes in a cell

DNA sequencing

• Sequencing projects have read the genomes of a range of organisms (e.g. The Human
Genome Project)
• Sequencing methods are continuously updated and have become automated
• Past – labour-intensive, expensive, could only be done on a small scale
• Now – automated, cost-effective and done on a large scale e.g. pyrosequencing

Applications of sequencing projects

Simple organisms
• Determining the genome of simpler organisms allows the assignment of proteins to each
gene in the genome (proteome), creating a database
• Easy because less non-coding DNA
• Identifying the protein antigens on the surface of viruses / pathogenic bacteria can help
in the development of vaccines

Complex organisms e.g. humans

• Knowledge of the genome cannot easily be translated into the proteome, due to the
presence of:
• Non-coding DNA
• Regulatory genes – determine when the genes that code for particular proteins
should be switched on and off
• Human Genome Project – determined the sequence of bases of a human genome

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3.8.4 Gene technologies allow the study and alteration of gene

function allowing a better understanding of organism function and
the design of new industrial and medical processes

[Link] Recombinant DNA technology

Recombinant DNA technology

• The transfer of DNA fragments from one organism or species, to another

• Transferred DNA can be transcribed / translated into proteins within cells of the
recipient (transgenic) organism
• Since the genetic code is universal
• As are transcription and translation mechanisms

Fragments of DNA can be produced by several methods, including:

Method 1 - Conversion of mRNA to complementary DNA (cDNA), using reverse

transcriptase

1. mRNA isolated from a cell that readily synthesises the protein coded for by the desired
gene
2. Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses
mRNA as a template to synthesise a single strand of cDNA
3. DNA polymerase forms second strand of DNA (= double stranded = gene) using cDNA as
a template

Method 2 - Using restriction enzymes to cut a fragment containing the desired gene from
DNA

1. Different restriction endonucleases cut DNA at

specific sequences of bases called a ‘recognition
sequence’
• Shape of recognition site complementary
to active site
2. Some restriction enzymes cut in a staggered
fashion = ‘sticky ends’ formed (uneven cut is left in
which each strand of DNA has exposed, unpaired
bases)

Method 3 - Creating the gene in a ‘gene machine’

• Synthesises fragments of DNA from scratch without the need for a pre-existing DNA
template
• DNA fragments produced quickly / accurately
• Free of introns → can be transcribed by a prokaryote who can’t remove introns

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Advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut
gene from DNA

• More mRNA in cell than DNA → easily extracted

• Introns removed by splicing (in eukaryotes) whereas DNA contains introns
• Bacteria can’t remove introns

The culture of transformed host cells as an in vivo method to amplify DNA fragments

Promotor and terminator regions added to fragments of DNA

• Promotor and terminator regions need to be added in order for the gene to be
transcribed and then translated into a protein
• Promotor regions – DNA sequences that tell RNA polymerase when to start
producing mRNA
• Terminator regions – tell RNA polymerase when to stop

The use of restriction endonucleases and ligases to insert fragments of DNA into vectors
• Vector transports DNA into host cell e.g. plasmids or bacteriophages
• Vector DNA and DNA fragment cut using same restriction enzyme
• Vector DNA and DNA fragment have complementary sticky ends →
complementary base pair
• DNA ligase forms phosphodiester bond between adjacent nucleotides on sticky ends

Transformation of host cells using these vectors

• Host cells have to be persuaded to take in the plasmid vector and its DNA
• e.g. host cells placed into an ice-cold calcium chloride solution to make cell walls
more permeable, then plasmids added, and mixture is heat shocked
• Bacteriophage vector – infects host bacterium by injecting its DNA into it, then the
phage DNA with target gene integrates into bacterial DNA

The use of marker genes to detect genetically modified (GM) cells / organisms
• Not all cells / organisms will take up the vector and be transformed
• Marker genes, inserted into vectors at same time as target gene, are added in order to
identify which cells have the desired gene
• Gene markers can be:
• Resistance to an antibiotic
• Fluorescent protein
• Enzyme whose action can be identified

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The principles of the polymerase chain reaction (PCR) as an in vitro method to amplify
DNA fragments

PCR: amplification specific DNA fragments (in every cycle, amount of DNA doubles)

Reaction mixture: DNA fragment + DNA polymerase (taq polymerase) + (forward/reverse)

primers + nucleotides

Cycle repeated 30-40 times to make lots of copies of DNA fragment:

Step 1: strand separation (95oC)

• Separates DNA strands by breaking
hydrogen bonds between bases

Step 2: primer annealing (55oC)

• Allows primers to bind / anneal to DNA
fragment template strand, forming hydrogen bonds
• Primer = short, single stranded DNA fragment
• Primer complementary to template DNA at edges of region to be copied
• DNA polymerase binds to primer to start synthesis; it can only add nucleotides
onto pre-existing 3’ end
• Two different primers required (forward and reverse)
• Because DNA strands run in anti-parallel, but polymerase can only run in
one direction

Step 3: DNA strand synthesis (72oC)

• Optimum temperature for DNA polymerase to make complementary copies of DNA
• Nucleotides align next to complementary exposed bases
• DNA polymerase joins adjacent nucleotides, forming phosphodiester bonds

You should be able to evaluate the ethical, financial and social issues associated with the
use and ownership of recombinant DNA technology in agriculture, in industry and in
medicine

You should be able to balance the humanitarian aspects of recombinant DNA technology
with the opposition from environmentalists and anti-globalisation activists

You should be able to relate recombinant DNA technology to gene therapy

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