Bioveterinary Sciences &
Biological Sciences Notes
Royal Veterinary College, D300
Contents
INHERITANCE, GENETICS & EVOLUTION
Cell and Molecular Genetics ..................................................................................................... 1
Protein Structure ............................................................................................................................ 1
Enzymes .......................................................................................................................................... 5
Nucleic Acids ................................................................................................................................. 11
Genes, Chromatin, and Chromosomes ......................................................................................... 14
Transcription ................................................................................................................................. 19
Translation .................................................................................................................................... 25
Protein Sorting and Secretion ....................................................................................................... 30
Manipulating Genes ............................................................................................................... 34
Polymerase Chain Reaction .......................................................................................................... 34
Recombinant DNA Technology ..................................................................................................... 41
Sequencing DNA ........................................................................................................................... 45
Transgenic Manipulation .............................................................................................................. 52
Genetics of Animals and Populations ...................................................................................... 63
Bioinformatics ............................................................................................................................... 63
Introduction to Animal Genetics................................................................................................... 69
Meiosis .......................................................................................................................................... 80
Animal Genetics in Livestock and Companion Animals ................................................................ 88
Introduction to Evolution ....................................................................................................... 94
Darwinian Mechanisms of Evolution ............................................................................................ 94
Philosophy and History of Pre-Evolution ...................................................................................... 97
Origins of Life .............................................................................................................................. 102
Mendelian Genetics and Evolution ............................................................................................. 112
Species, Variation, and Speciation .............................................................................................. 118
Evolution and Development ....................................................................................................... 122
Domestication and Selection of Productive Traits...................................................................... 125
The Modern Synthesis of Evolution ............................................................................................ 127
, 1
Protein Structure
The basics of protein structure.
Structural elements and the forces that determine them.
Structure affecting function.
The basic building blocks of mammalian proteins are α-amino acids. The linear arrangement of α-
amino acids within a polypeptide chain determines protein primary structure. Protein structure
determines function.
Peptide bonds can form between the N-
terminus of one amino acid and the C-
terminus of another amino acids. The
reaction is catalysed by peptidyl
transferase (a 28S ribozyme – part of the
60S ribosomal subunit).
The peptide bond is planar.
Covalent bonds maintain primary
structure.
- Bond strengths ~100 kcal/mole
Hydrogen bonds maintain secondary structure.
- Bond strengths ~3-7 kcal/mole
o In α-helices, hydrogen bonds run within the chains
(hydrogen bonds run parallel to the helix axis).
o In β-sheets, hydrogen bonds form between adjacent
sheets (hydrogen bonds run perpendicular to chain
direction).
Hydrophobic and van der Waals bonds maintain tertiary structure.
- Bond strengths ~1-2 kcal/mole
Electrostatic bonds found within the protein interior can help to maintain tertiary structure and are
often associated with mechanism.
- Bond strengths ~3-7 kcal/mole
Van der Waals and electrostatic bonds are the principal bonds required for quaternary structure.
, 2
Within a polypeptide chain, individual amino acids are refer to as residues. When >50 amino acids
are present, a polypeptide is referred to as a protein (M.W. ≥6000kDa).
α-helix – where the α-carbon backbone winds around an axis such that
each carbonyl oxygen atom is hydrogen-bonded to each amide nitrogen
of the amino acid located 4 residues closer to the C-terminus.
Α-helix is stabilised by hydrogen bonding between NH and C=O of main
chain. All the C=O and NH groups hydrogen bonded for α-helix.
Hydrogen bonding is four residues ahead in linear sequence. The helix
can be right handed (clockwise, like in most proteins) or left handed
(counter clockwise).
Proline and glycine will interfere with the α-helix.
R groups are pointed outward but are not used in the helix structure.
The R groups influence the way protein interacts with other proteins
and the environment.
β-sheet – there are two kinds of β-sheets. Both fully utilise the
hydrogen bonding potential of the peptide bond.
- Parallel β-sheets
- Antiparallel β-sheets
Amino acid R groups can be:
- Non polar, e.g. glycine, alanine, valine
- Polar, e.g. serine, tyrosine, glutamine
- Electrically charged, e.g. aspartic acid and glutamic acid (acidic), lysine and arginine (basic)
, 3
The arrangement of the primary and secondary structural elements in three dimensional space
constitutes the tertiary structure of a protein – i.e. the conformation of a protein. This is a
characteristic feature of a protein at a given temperature and pH.
A domain is the smallest stable unit of tertiary structure. A domain is defined as that region of a
polypeptide chain that can fold into an autonomous stable tertiary structure. Domains are stable
because they are independent structural units – but they also represent independent functional
units. Throughout evolution domains have been swapped between proteins (domain shuffling).
Proteins that possess quaternary structure are composed of two or more subunits.
- Haemoglobin – a carrier protein, transporting O2 or CO2 around the body. It is made up from 2
pairs of different polypeptide chains.
- Collagen – a structural protein giving strength and resilience to skin. It is made up from three
separate γ-helical strands.
Small changes in oxygen concentration can dramatically affect its binding to haemoglobin. This gives
a sigmoidal curve of oxygen binding.
, 4
Collagen
Collagen is an extracellular fibrous protein – but it
is not an α-helix. Collagen is composed of three
helical chains that wind around a central axis – a
triple γ-helix.
The general structure is: Gly-X-Y, where:
- X can be any amino acid (but especially
proline, lysine or hydroxylysine).
- Y is typically always proline or hydroxyproline.
- Gly is glycine.
Because glycine is small, the glycines from each chain fit at the centre of each helix. Hydrogen bonds
between chains link all 3 helical chains together.
P53
p53, also known as TP53 or tumour protein is a gene
that codes for a protein that regulates the cell cycle
and hence functions as a tumour suppression. It is a
transcription factor and it’s very important for cells in
multicellular organisms to suppress cancer. P53 has
been described as "the guardian of the genome",
referring to its role in conserving stability by
preventing genome mutation. It is one of the most
mutated genes that leads to cancer formation. The
name is due to its molecular mass: it is in the 53
kilodalton fraction of cell proteins.
, 5
Enzymes
Thermodynamics of Reactions
Define the following terms: substrate; product, reaction rate; equilibrium
constant; rate constant; activation energy; induced fit.
Outline the physical basis of how enzymes achieve massive reaction rate
increases as compared with uncatalyzed reactions.
kF
Substrate Product
kR
𝑘
𝐸𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡, 𝐾 =
𝑘
kF Transition kF
Substrate Product
kR State kR
Any chemical reaction passes through a transition state in which old bonds are incompletely broken
an new bonds are incompletely formed. The transition state has a different geometry from either
the reactants or products. For example, in the case of phosphoryl transfers, catalysed by kinases, a
novel pentavalent structure (trigonal bipyramid) has a transient existence:
Enzymes act as catalysts and affect energetics of transition state. They don’t affect the energetics of
the reactant or product, just the transition state
intermediate. All enzymes lower the transition state
energy of a reaction through:
- Proximity: Enzymes bring substrates together.
Increases rate by 105 - 108.
- Orientation: Enzyme orients substrates so they
best attain the most favourable position to
achieve transition state. Increases rate by 104.
- Microenvironment: Active site can provide
protected, non-aqueous environment (e.g. for a
species that’s hydrophobic but when the
reaction takes place in an aqueous
environment). Increases rate by 104.
, 6
Enzymes catalyse only one or a limited number of chemical reactions. Catalysis is achieved
by amino acid residues brought close together in the tertiary structure (which can be
separated in the primary structure) to form an active site. The optimal conformation of the
binding site is one that precisely contours the transition state of the reactants.
The active site is not a rigid structure. It moulds itself around the substrate for the reaction
which defines the induced fit model of enzyme action where the enzyme changes
conformation upon substrate binding.
Each enzyme displays a characteristic pH profile. The optimum pH often reflects pH-
sensitive events at the active site. This relates to the R-groups in the amino acids.
, 7
Michaelis-Menten Kinetics
Explain in qualitative terms using a graphical representation of enzyme activity
plotted against substrate concentrations.
k+1 k+2
k-1
E = Enzyme
S = Substrate
ES = Enzyme-Substrate Complex
The enzyme, E, catalyses the conversion of S to yield a product P.
k+1, k-1 are the rate constants for the forward and backward binding step.
k+2 is the catalytic rate constant for the reaction.
Enzymes act by increasing the rates of both the forward and backward reactions equally. They do
not change the equilibrium constant (K).
Hence, the equilibrium constant, K (i.e. = [S]/[P]), remains unchanged and determines the forward or
reverse reaction. We can measure the activity by looking at the [product] or [reactant] over time.
Single subunit enzymes displays characteristic profiles when their activity is plotted against
increasing substrate concentrations. Such a profile is said to be hyperbolic. Vmax is limited by the
amount of enzyme present.
, 8
Regulation of Enzyme Activity
Outline the properties of an allosteric enzyme and explain qualitatively how its
action differs from enzymes exhibiting Michaelis-Menten kinetics.
Outline the control of enzymic activity by: phosphorylation/dephosphorylation;
protein and/or nucleotide binding; Ca 2 + binding in EF-hand; proteolysis.
Enzymes rarely operate alone; they are part of a chain of reactions, known as a pathway e.g.
metabolic pathways. This is to control the amount of products formed as some compounds can be
toxic in large amounts.
Many metabolic pathways are controlled by
feedback mechanisms, e.g. E may inhibit the
conversion of C to E. Likewise, B may activate the
conversion of C to D and d to E.
Allosteric enzymes, close to the beginning, end, or at
branch points of a metabolic pathways, are able to
bind products of remote reactions in the same or
related pathway. They do this at separate ‘allosteric’
sites, distinct from the active
site of that enzyme.
Allosteric enzymes have
quaternary structure (they
are multi-subunit enzymes),
and display S-shaped kinetic
profiles i.e. show
cooperativity. Such curves
can be changed rapidly by
activators or inhibitors, each
binding to different sites on
the enzyme.
- Allosteric inhibitors can be viewed as stabilising the T
state. This decreases the affinity of the enzyme for
substrate.
- Allosteric activators can be viewed as stabilising the R
state. This increases the affinity of the enzyme for
substrate.
Both activators and inhibitors bind to sites on each subunit
remote from the active site.
, 9
Effect of Activators and Inhibitors on S-shaped Kinetic Profiles
Activator shifts the curve to the left. Inhibitor shifts the curve to the right.
A protein can exist in one of two symmetrical states: R & T:
- In the R form, the enzyme has a high affinity for
substrate.
- In the T form, the enzyme has a low affinity for
substrate.
Binding of the first substrate molecule, S, to the T state is
unfavourable – but once it occurs both subunits flip to the
R state, which has a higher affinity for substrate.
The regulation of enzyme activity can occur through several
different mechanisms involving enzyme modification. These
can be in addition to, or instead of, allosteric mechanisms.
1. Control by covalent modification, especially phosphorylation.
By far the most common form of covalent modification used to control enzyme activity is
phosphorylation/dephosphorylation.
- Enzymes that bring about phosphorylation are known as kinases.
- Enzymes that cause dephosphorylation are known as phosphatases.
There are two forms of kinase: serine / threonine kinases and tyrosine kinases.
Phosphorylation reactions usually require ATP as phosphate donor, producing ADP in
addition to covalently linking a phosphate group to serine, threonine or tyrosine on the
target protein.
