TOPIC 1: ATOMIC STRUCTURE AND CHEMICAL BONDS

Atoms

• Atoms are the building blocks of all matter in the Universe.

• Each atom has three basic parts namely: electrons, protons, and neutrons.

• Electrons (negatively charged) are the smallest of the three and are found in orbitals
within shells.

• At the centre of the atom is the nucleus that usually consists of an equal number of
protons (positively charged) and neutrons (no charge).

• The mass of the atom is the sum of the number of protons and neutrons.

• The hydrogen atom (atomic mass = 1unit) contains 1 proton and one electron (zero mass)
and no neutrons.

• The number of protons is equal to the number of electrons for any given atom.

• The structure and functions of biological molecules are determined by the structure and
behaviour of the constituent atoms.

Orbitals and shells

• An orbital is the physical region or space where an electron is expected to be present.

Any given orbital can be occupied by a maximum of two electrons.

• Orbitals exist at different energy levels with the lowest level being the ground state.

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 • The orbital with the lowest energy is the s orbital (represented by sharp spectroscopic
lines).

• The next orbital is the p (represented by principle spectroscopic lines).

• Orbitals are arranged in form of shells in which the innermost shell has one orbital ( 1s )
which can be occupied by a maximum of two electrons.

• The second shell has four orbitals (one 2s orbital and three 2p orbitals).

o The three 2p orbitals are 2px, 2py and 2pz and each one can be occupied by a
maximum of two elctrons.

o Therefore the second shell can be occupied by a maximum of eight electrons.

• The third shell has four orbitals (one 3s, and three p orbitals), etc.

Atomic structure and electron configuration of some elements

• Figures 1.1 and 1.2 show the atomic structures and electron configurations of some
elements which are important constituents of biomolecules.

(a) Hydrogen (b) Carbon (c) Oxygen

Figure 1.1. Atomic structures of the hydrogen (a), carbon (b) and oxygen (c).

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 (a) Nitrogen (b) Phosphorus (c) Sodium

(d) Chlorine
Figure 1.2. Atomic structures of the nitrogen (a), phosphorus (b) sodium (c) and chlorine
(d).

Chemical bond

• A chemical bond is an attraction between atoms and it results into the formation of a
chemical compound (or molecule) that contains two or more atoms.

• A chemical bond is caused by the electrostatic force of attraction between opposite

charges, either between electrons and protons (in the nucleus), or as the result of a dipole
attraction between neighbouring particles.

• There are two major classes of chemical bonds i.e. (i) covalent and (ii) non-covalent
bonds.

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Covalent bond

• A covalent chemical bond results from the equal sharing of electrons between two atoms.

• A single covalent bond represents the sharing of two valence electrons, usually from two
atoms of different elements but at times from atoms of the same element.

• The hydrogen molecule (H2) has a single covalent bond in which two electrons are shared
equally between the two hydrogen atoms (H: H or H-H).

• In methane (CH4), each of the four CH bonds is a single covalent bond (C: H or C-H) in
which each hydrogen atom shares one pair of electrons with a common carbon atom
(Figure 1.3).

• Multiple covalent bonds are common for certain atoms. For example, ethylene (C2H4)
has a double covalent bond (H2C=CH2) which results from the sharing of two pairs of
electrons between the two carbon atoms (Fig 1.3).

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 • The nitrogen molecule (N2) has a triple covalent bond between the two nitrogen atoms
(Fig 1.3).

• Covalent bonds are very stable because the energies required to break them are much
greater than the thermal energy available at room temperature (25 ºC) or human body
temperature (37 ºC).

(a) Methane (b) Ethene (c) Nitrogen

Figure 1. 3. Four single C-H bonds in methane (a), a double C=C bond in ethene (b) and a
triple N≡N bond in molecular nitrogen.

• In molecular nitrogen, three pairs of electrons are shared by the two nitrogen atoms while
the fourth pair of each nitrogen atom is unshared (lone pair).

Noncovalent interactions

• The four main types of noncovalent interactions that occur in biological systems are:
o (i) ionic bonds
o (ii) hydrogen bonds
o (iii) van der Waals interactions
o (iv) hydrophobic interactions.

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Ionic bond

• An ionic bond is formed by the attraction of oppositely charged atoms or groups of

atoms.

• When an atom gains or loses one or more electrons, it forms an ion.

• Ions have either a net positive or net negative charge.

• Positively charged ions are attracted to the negatively charged 'cathode' in an electric
field and are called cations. They result when an atom loses one or more electrons.

• Anions are negatively charged ions which are attracted to the positively charged 'anode'
in an electric field. They result when an atom gains one or more electrons.

• Every ionic chemical bond is made up of at least one cation and one anion.

• Sodium (atomic number 11) forms an ionic bond with chlorine (atomic number 17) in
sodium chloride (common salt) in which sodium donates its electron to chlorine and the
two coexist as sodium ion (cation) and chloride ion (anion). See Fig 1.4.

Figure 1.4. An ionic bond formed between a sodium ion (cation) and a chloride ion
(anion) in the formation of sodium chloride (common salt).

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Hydrogen bonds

• A hydrogen bond is the interaction of a partially positively charged hydrogen atom in a

polar molecular (e.g., water) with unpaired electrons from another atom, either in the
same molecule (intramolecular hydrogen bond) or in a different molecule (intermolecular
hydrogen bond). See Fig 1.5.

• Normally, a hydrogen atom forms a noncovalent bond with only one other atom.

(a) (b) (c)

Figure 1.5. Hydrogen bonds: (a) between different water molecules, (b) between
water and a peptide and (c) between water and a carboxyl group .

• In liquid water, each water molecule forms temporary hydrogen bonds with several
others, creating a dynamic network of hydrogen-bonded molecules (Fig 1.5a). Water also
can form hydrogen bonds with peptide and carboxyl groups and this explains the high
solubility of polypeptides and organic acids (Figs 1.5 b & c). The peptide and ester
groups, which are present in many biomolecules, commonly form hydrogen bonds with
water making them highly soluble.

Van der Waals interactions

• When any two atoms approach each other closely, they create a weak, nonspecific
attractive force called a van der Waals interaction.

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 • Such nonspecific interactions result from the temporary and random fluctuations in the
distribution of the electrons of any atom, which give rises to a temporary unequal
distribution of electrons.

• The van der Waals interactions are responsible for the attraction between molecules of
nonpolar liquids and solids, such as long chain biological molecules that cannot form
hydrogen bonds or ionic interactions with other molecules.

Hydrophobic interactions

• Nonpolar molecules or nonpolar portions of molecules tend to aggregate in water due to a

phenomenon called the hydrophobic effect.
• In an aqueous environment, nonpolar molecules aggregate with their hydrophobic
surfaces facing each other and because of this, less water is needed to completely
surround the nonpolar molecules.
• Therefore, water forces the nonpolar molecules to spontaneously form aggregates.
• In addition, it is observed nonpolar molecules dissolve in nonpolar solvents because of
the hydrophobic interactions between the nonpolar molecules and the nonpolar solvents.

Formation of covalent bonds in water

In a water molecule (H2O), two hydrogen atoms each share their one electron with oxygen to
form two covalent bonds .
• Oxygen forms one single covalent with each hydrogen.
• The structural formula of a water molecule is written as given in Fig 1.6.

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 Figure 1.6. Chemical bonds in a water molecule. A single bond forms between an
oxygen atom and each of the two hydrogen atoms.

Formation of covalent bonds in carbon dioxide

• In the carbon dioxide molecule (CO2), two oxygen atoms and one carbon atom will each
share two electrons to form four covalent bonds.

• The carbon atom has two covalent bonds with each oxygen atom.

• Each covalent bond between carbon and one oxygen is called a double bond.

• The structural formula of a carbon dioxide molecule is written as given in Fig 1.7.

Figure 1.7. Chemical bonds in a water molecule. A double bond forms between a
carbon atom and each of the two oxygen atoms.

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Revision exercise

1. Describe the general structure of an atom.

2. Describe the distribution of electrons in atomic orbitals and atomic shells.
2. Differentiate between an atomic shell and an atomic orbital.
3. Draw the atomic structures of hydrogen, carbon, oxygen, nitrogen, phosphorus, sulphur,
sodium and chlorine.
4. Describe the nature of covalent and non-covalent chemical bonds.

Summary
• Atoms are the building blocks of all matter in the Universe.
• Each atom has three basic parts namely: electrons, protons, and neutrons.
• Electrons (negatively charged) are the smallest of the three and are found in shells or
orbitals.
• At the centre of the atom is the nucleus that consists of an equal number of protons
(positively charged) and neutrons (no charge).
• The mass of the atom is determined by the number of protons and neutrons i.e. protons +
neutrons.
• The hydrogen atom (whose atomic mass = 1) contains 1 proton and one electron but does
not contain any neutrons.
• The number of protons is equal to the number of electrons for any given atom.
• Electrons are found in different orbitals around the nucleus.
• The integrity and functions of biologically important molecules are determined by the
structure and behaviour of the constituent atoms.

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2. NUCLEIC ACIDS

2.1 INTRODUCTION
• Nucleic acids are large biomolecules which store, transmit and express genetic
information of a cell.

• They are essential for life because they constitute the genetic material of all living
organisms.

• The two types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA).

• DNA is the store of genetic information which is transmitted from one generation to
the next in most cells.

• RNA is normally used as an intermediate in the transfer of information from DNA to

proteins but in some viruses RNA is also the store of genetic information.

• Nucleic acids are made up of units called nucleotides which are arranged in extremely
long molecules known as polynucleotides.

2.2 NUCLEOTIDE
• A nucleotide has three components:

i. A 5- carbon (pentose) sugar which is either ribose or deoxyribose.

▪ The OH on C3 of the forms the three-prime (3ꞌ) end

▪ The OH on C5 forms the five-prime (3ꞌ) end

ii. A nitrogenous base which can either be:

▪ a two ring purine (adenine-A or guanine-G)

▪ or a one ring pyrimidine (cytosine-C, thymine-T or uracil-U).

▪ The nitrogenous base T is only found in DNA while U is only

found in RNA

iii. A phosphoric acid.

Figure 2.1. Ribose and deoxyribose sugars.

Figure 2.2. The two types of nitrogenous bases found in DNA and RNA

Figure 2.3 The two purine bases found in DNA and RNA

Figure 2.4 The three pyrimidine bases found in DNA (C and T) and RNA (C and U)
Figure 2.5 Phosphoric acid gives the nucleic acids their acidic character.

2.2.1 Formation of a nucleoside

• A nucleoside is formed through a condensation reaction resulting in a beta covalent
glycosidic bond between a pentose sugar and a nitrogenous base.

• The OH group of carbon 1 (C1) of the pentose combines with the H at position 1 of a
pyrimidine base to form a pyrimidine nucleoside

• The OH group of C1 of the pentose combines with the H at position 9 of a purine to

form a purine nucleoside.
Figure 2.6 Formation of a different nucleosides by condensation.

2.2.3 Formation of a nucleotide

• A nucleotide is formed through a phosphoester bond between a nucleoside and a
phosphate (or phosphoric acid).

• The OH group of carbon 5 of the pentose sugar of the nucleoside combines with H of
the phosphate in a condensation reaction.

Figure 2.7 Formation of a nucleotide (guanosine monophosphate) by condensation.

Figure 2.8 Diagrammatic representation of nucleotide formation
Table 2.1 Summary of the formation of the nucleotides of DNA and RNA
Base Nucleoside Nucleotide RNA DNA Symbol
Adenine Adenosine Adenosine monophosphate AMP dAMP A

Guanine Guanosine Guanosine monophosphate GMP dGMP G

Cytosine Cytidine Cytidine monophosphate CMP dCMP C
Thymine Thymidine Thymidine monophosphate TMP dTMP T
Uracil Uridine Uridine monophosphate UMP dUMP U
2.2.4 Formation of a dinulceotide

• A dinucleotide is formed in a condensation reaction between the OH of carbon 3 of

the pentose sugar of the first nulceotide and H of the phosphate on carbon 5 of the
second nucleotide resulting in a bond called a phosphodiester bridge.

Figure 2.9 Diagrammatic representation of dinucleotide formation

Figure 2.10 Chemical reaction during the formation of a dinucleotide of RNA

2.2.5 Formation of DNA and RNA polynucleotides
• The dinucleotide is converted to a trinucleotide by addition of a nucleotide to the OH
on carbon 3 of the sugar of the second nucleotide.

• The addition of more nucleotides continues at the 3′ end of the growing chain until the
required length of a DNA or RNA strand is produced.

2.3 DNA STRUCTURE AND FUNCTION

• The DNA molecule is made of two strands and is therefore said to be double stranded.

• Each strand is a polymer of the four types of nucleotide bases (A, G, C and T)

2.3.1 Chemical properties of DNA

• DNA is acidic in nature because it’s rich in phosphates.

• Erwin Chargaff (1949) did some chemical analysis of DNA and came up with
findings known as Chargaff’s rules which state that:

o the total number of purine bases (A+G) = the total number of pyrimidine bases
(C+T)

o The number of adenine bases = the number of thymine bases (i.e. the ratio A:
T = 1)

o The number of guanine bases = the number of cytosine bases (i.e. the ratio G:
C = 1).
2.3.2 Physical properties of DNA
• Maurice Wilkins and Rosalind Franklin passed X-rays through DNA fibres (X-ray
crystallography) and found that:
o DNA had a regularly twisted (helical) structure
o There was a regular (repeating) spacing of 3.4nm between the components of
DNA polymer and that the diameter of each DNA polymer was 20nm
o DNA was made of two strands.
2.3.3 The Watson - Crick Model of DNA structure
• Watson and Crick (1953) used X-ray crystallography results from Wilkins and
Franklin as well as Chargaff’s rules to work out their model of DNA.

• Using pieces of wire and flat metal shapes, they concluded that DNA was in form of a
double helix composed of two polynucleotide chains held together by hydrogen
bonding between pairs of bases.

• They thought of DNA as similar to a ladder in which the base pairs are the steps and
the sugar-phosphate backbones are the two sides.

• The backbone is then twisted into a double helix in which there are ten nucleotide
bases per turn. T

• hey proposed that the sequence of nucleotides in one strand determines the sequence
in the other meaning that the two strands are complementary and anti-parallel.

• In 1962 Watson and Crick together with Wilkins were awarded the Nobel Prize for
Medicine for elucidating the structure of DNA.

Figure 2.13 The DNA double helix (a) and double helix (b). Adenine (A) is paired with
thymine (T) while guanine (G) is paired with cytosine(C). The presence of thousands of
hydrogen bonds in a DNA molecule contributes greatly to the stability of the double helix.

2.4 RIBONUCLEIC ACID (RNA) STRUCTURE

• RNA is a single polynucleotide which is synthesised based on the information carried
by DNA.

• The three types of RNA are messenger, transfer and ribosomal RNAs.
 • RNA contains adenine, guanine, cytosine and uracil but not thymine.

• In eukaryotes, RNAs are synthesized in the nucleus.

o rRNA is synthesized in special region of the nucleus called the nucleolus.

2.4.1 Messenger RNA (mRNA)

• It is a single-stranded molecule formed on a single strand of DNA by transcription.

• It is formed according to the rules of complementary base-paring under the influence

of RNA polymerase.

• The base sequence of RNA is a template copy of the template DNA strand and varies
in length according to the length of the polypeptide that it codes for.

• The mRNA carries the genetic code (codon) according to the template DNA.

2.4.2 Ribosomal RNA (rRNA)

• It is normally a single-stranded nucleic acid.

• It is synthesised (transcribed) from the DNA located in the nucleolar organiser of

several chromosomes.

• It combines with protein to form ribosomes which are the site of proteinsynthesis.

• Ribosomes are usually found in clusters (polyribosomes) linked together by a strand

of mRNA.

• Each ribosomes is made up of two subunits ( the large and the small)

• The prokaryotic ribosome has a size of 70s ( 50s + 30s)

• The Eukaryotic ribosome has a size of 80s ( 60s + 40s)

2.4.3 Transfer RNA (tRNA)
• It is normally a single-stranded polynucleotide.

• It is an adaptor molecule which is involved in the transfer of amino acids from the
cytoplasm to the ribosomes during the process of proteinsynthesis.

• The tRNA has four arms

o the acceptor arm

o the variable arm

o the D arm

o the anticodon arm.

• Each amino acid has its own tRNA which carries a triplet anticodon for the particular
amino acid.

• About 60 different tRNA have been identified and all have the same basic structure.

• The 5´ end of tRNA always ends in the base sequence of CCA.

• The structure of tRNA is described by the clover leaf model.

Figure 2.14 Transfer RNA (tRNA). (a) Schematic diagram of tRNA for phenylalanine.
Table 2.2 : Similarities between DNA and RNA structure and function
DNA and RNA
1 Present in all prokaryotes and eukaryotes
2 Carry genetic information
3 Made up of nucleotides
4 Polymerization results in strand formation
5 Cleaved by nucleases

Table 2.3 : Differences between DNA and RNA structure and function
DNA RNA
1 Stores and transmits genetic information Mainly transmits genetic information
2 Mainly double stranded Mainly single stranded
3 Chemically stable Chemically unstable
4 Has thymine in the place of uracil Has uracil in the place of thymine
5 Huge molecule Relatively smaller in size
6 Usually one type Mainly three types (rRNA, tRNA and mRNA)
TOPIC 3: DNA REPLICATION

3.1 Introduction
DNA is a biomolecule which is capable of reproducing itself in a process called replication.
Watson and Crick proposed that during replication the two DNA strands were capable of
separating and acting as templates to which a complementary set of nucleotides would attach
by base pairing to form new DNA strands.

3.2 Objectives: At the end of this unit you should be able to:
(i) Prove that DNA is capable of accurately reproducing itself.
(ii) Explain the semi-conservative mechanism of DNA replication with reference to the
experiments of Meselson and Stahl.
(iii) List the enzymes and proteins that are involved in prokaryotic DNA replication and
explain
the role of each of them.
(iv) Explain the different stages of prokaryotic DNA replication
(v) Differentiate between the synthesis of the leading strand and that of the lagging strand

3.3 Semi-conservative mechanism of DNA replication: The replication of DNA is semi-

conservative in which the original double helix molecule is duplicated into two identical
copies. Each DNA copy contains one old strand and one new strand. Evidence of this
mechanism of DNA replication came from the experiment by Meselson and Stahl who used
E.coli cells.

The cells were initially grown in a nutrient medium containing ‘heavy’ nitrogen isotope (15N)
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for many generations until all the nitrogen in their DNA contained N. The cells were then
transferred to a medium containing the ‘light’ nitrogen isotope (14N) and different generations
of bacterial cells were sampled from the medium. The DNA in each sample was analysed by
density-gradient equilibrium centrifugation using caesium chloride (CsCl). In this method
different concentrations of CsCl were added to a centrifuge tube. The most concentrated
solution was added first followed by less concentrated solutions and the most dilute solution
was added last. This CsCl solution gradient is able to separate heavy-heavy (HH), light-light
(LL) and heavy-light (HL) DNA double helices into separate bands. The bands which were
observed in the experiment proved that DNA undergoes the semi-conservative replication
and not the conservative replication. The semi-conservative type of DNA replication has also
been conformed in eukaryotic cells
Figure 3.1.The Meselson-Stahl experiment. (a) Cells were grown for many generations in a medium containing
only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N as shown by a single band (blue) when
centrifuged in a CsCl density gradient. (b) Once the cells had been transferred to a medium containing only light
nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient
(purple band). (c) Continuation of replication for a second generation yielded two hybrid DNAs and two light
DNAs (red), confirming semi conservative replication. Adapted from Griffiths et al. 1993. An introduction to
Genetic Analysis. Page 316.

3.4 DNA replication process in prokaryotes

DNA replication in prokaryote is well represented by E.coli. The process involves the
following major steps: (i) Replication initiation, (ii) DNA denaturation – melting –unzipping,
(iii) Priming, (iv) Elongation - DNA synthesis at growing fork (v) Separation of circular
daughter molecules, (vi) Proof reading.
3.4.1 Initiation of replication in E.coli

The replication of DNA requires the removal of the supercoils and the attachment of enzymes
and other proteins to the origin of replication on the DNA molecule. The supercoils are
removed by the enzyme called topoisomerase followed by the attachment of a protein called
DnaA. This results in the formation of the initiation complex.

Figure 3.2. Initiation of DNA replication at E.coli origin of replication.

3.4.2 Double stranded DNA denaturation

This process is also called DNA melting or unzipping. When DnaA binds to the double helix,
the first separation of the two strands takes place. This process requires ATP. This is
followed by the binding of the enzyme DNA helicase which leads to the separation of more
base pairs. The helicase moves along the DNA double helix using the energy from ATP
hydrolysis to separate the two strands. The denaturation of double stranded DNA is in the 5′
→ 3′ direction. The single strand binding protein prevents the double helix from rejoining.
Figure 3.3. DNA denaturation by the action of helicase in E.coli.

3.4.3 Formation of RNA primers in E.coli

This process is called priming. It is well known that DNA polymerase cannot initiate a new
DNA chain but can only elongate a pre-existing DNA or RNA strand. Therefore, before the
parental DNA is replicated, a short RNA molecule, complementary the DNA strand, is
synthesised by RNA polymerase. This RNA molecule is called a primer and it attaches itself
to the initiation complex. A primer is synthesised on each DNA strand in the 5′→3′ direction.

Figure 3.4. Priming on the leading and lagging strand DNA templates by the action of
primase in E.coli.
3.4.4 DNA synthesis - addition of nucleotides at the growing fork by E. coli DNA
This is a polymerization reaction catalysed by DNA polymerase. Although the two strands of
DNA double helix are anti-parallel, DNA polymerase catalyses nucleotide addition at the 3’-
hydroxyl end of each growing chain. Therefore each strand is elongated only in the 5′→3′
direction. Synthesis of a new DNA strand involves the addition of deoxyribonucleotide
triphosphates (dNTPs) which include: dATP, dGTP, dTTP, and dCTP.

3.4.4.1 Leading strand synthesis: At each growing fork, one DNA strand, called the leading
strand, is synthesized continuously from a single primer on the leading strand template. The
leading strand grows in the 5’→3’ direction, like the growing fork.

3.4.4.2 Lagging strand synthesis: Synthesis of the lagging strand is more complicated,
because DNA polymerases can add nucleotides only to the 3’ end of a primer or growing
DNA strand. Movement of the growing fork exposes the lagging-strand template on which
short RNA primers are copied. Each of these primers is then elongated by addition of dNTPs
to its 3′ end. . In E.coli, this reaction is catalysed by DNA polymerase III (poly III). The
resulting short fragments containing RNA covalently linked to DNA are called Okazaki
fragments. In bacteria and bacteriophage, Okazaki fragments contain 1000-2000 nucleotides.
The overall direction of growth of the lagging strand is from its 3′→5′ end, complementary to
the polarity of its template but opposite to the direction of nucleotide addition by DNA
polymerases. DNA polymerase I is primarily involved in removing RNA primers from
Okazaki fragments (exonuclease activity) and filling the resultant gaps using its 5′→3′
polymerizing activity. DNA ligase joins adjacent completed Okazaki fragments.

DNA polymerase II functions in the inducible SOS response and also fills gaps and appears
to facilitate DNA synthesis directed by damaged templates. DNA polymerase II resembles
DNA polymerase I in its activity, but is a DNA repair enzyme, bringing about the growth in
5′→3′ direction using free 3′- OH groups.
Figure 3.5. DNA replication at a growing fork. Source: Lodish et al. 2000. Molecular Cell
Biology, 4th edition. Page 113.

3.4.5 Proof reading by DNA polymerase

Occasionally, a wrong base is inserted during DNA synthesis. This is corrected by the
proofreading function of all bacterial polymerases.

3.4.6 Separation of circular daughter molecules

During DNA replication the parental strands remain intact and retain their superhelicity. This
poses stearic and topological constraints to the completion of the replication of a circular
DNA molecule as the two growing forks approach each other. In E.coli, decatenation of the
two daughter cells is catalyzed by Topo II (DNA gyrase) and topoisomerase IV (Topo IV).
This helps separate the daughter DNA molecules from each other
Table 3.1 Enzymes involved in prokaryotic DNA replication
# Enzyme Function
1 DnaA protein Binds DNA to form the initiation complex
2 Topoisomerase Removes coils and supercoils from DNA
3 DNA polymerase I Removes primers from the new DNA strand
4 DNA polymerase II Repairs mistakes during DNA polymerization and also fills in
the gaps left after the removal of primers from lagging strand
5 DNA polymerase III DNA polymerization
6 Helicase DNA denaturation
7 Primase Synthesis of short RNA primers on template DNA
8 Ligase Joins DNA nucleotides through covalent bonds
9 Single strand binding protein Prevents single stranded DNA from forming double strands

3.5 Revision questions: DNA replication

1. Outline the central dogma of molecular biology.

2. Explain the experiments which provided evidence in support of the mechanism of DNA
replication.
3. Differentiate between conservative and semi-conservative mechanisms of DNA
replication.
4. Describe the various stages of prokaryotic DNA replication.
5. List the enzymes and non-enzyme proteins involved in prokaryotic DNA synthesis and
explain the role of each of them.
6. Differentiate between leading and lagging strand synthesis of prokaryotic DNA.
7. Explain how accuracy is achieved during DNA replication.

3.6 Summary: DNA replication

The DNA molecule is able to replicate itself and this is the basis of growth and reproduction among
living organisms. DNA replicates semi-conservatively with the leading strand template
replicating continuously and the lagging strand template replicating discontinuously. The
steps involved in DNA replication are (i) Replication initiation, (ii) DNA denaturation –
melting –unzipping, (iii) Priming, (iv) Elongation - DNA synthesis at growing fork (v)
Separation of circular daughter molecules, (vi) Proof reading. All these steps are controlled
by specific enzymes.
TOPIC 4: TRANSCRIPTION

4.1 Introduction

Transcription is the synthesis of RNA using information from DNA. This process ensures that
synthesis of proteins using information from DNA is properly regulated. Because of this
intermediary process, proteins are synthesised in the right place in the right amounts at the right
time.

4.2 Objectives: At the end of this topic you should be able to:
1. To explain the process of transcription.
2. To describe the phases of initiation, elongation and termination.
3. To discuss posttranscriptional modification of eukaryotic mRNA, tRNA and rRNA.

4.3 Mechanism of transcription

Transcription involves the following stages: (i) initiation (ii) elongation (iii) termination and (iv)
the processing of primary transcript products into mature RNA molecules.
4.3.1 Initiation
Before transcription can take place, the double helix in the gene to be transcribed has to unwind
and the two DNA polynucleotide chains have to separate (unzip) in order to expose the
nucleotide bases. This is done with the help of the enzyme helicase and other proteins. This is
followed by the binding of RNA polymerase to the unzipped part of DNA at a region called
promoter. During the initiation phase, the first two nucleotides of the RNA (usually A and C) are
joined together.

Figure 4.1. Initiation of RNA transcription

4.3.2 Elongation

The elongation phase begins when the RNA polymerase moves along the sense DNA strand
binding one nucleotide at a time as it moves. RNA polymerase adds nucleotides to the 3′- end,
building the new RNA in the 5′ → 3′ direction and opening the DNA chain as it moves along.

Figure 4.2. Elongation during RNA synthesis/transcription

4.3.3 Termination of transcription

There are two types of transcription termination mechanisms.

(i) The first mechanism which is referred to as rho (ρ)-independent termination of
transcription, occurs at specific base sequences called palindromic sequences in the
RNA molecule (AAAGGCUCC-UUUU-GGAGCCUUU). When the palindromic
sequence is transcribed, it forms a hair-pin loop which disturbs the RNA polymerase and
results in termination of transcription;

Figure 4.3 Rho-independent termination of transcription

 (ii) the second mechanism is the (rho) ρ-dependent termination which uses the ρ factor to
stop RNA synthesis at specific sites. When the RNA polymerase reaches certain sites of
DNA the rho factor approaches the new mRNA and hooks it up thereby disturbing the
RNA polymerase and causing the termination of the transcription process.

4.3.4 Posttranscriptional modification of newly synthesized RNAs

The newly synthesized RNAs (mRNA, tRNA and rRNA) are called precursor molecules and
must be modified before they become mature and functional. Modification involves removal of
some nucleotides and transformation of other nucleotides.
4.3.4.1 Messenger RNA posttranscriptional modification
Precursor mRNA undergoes capping, polyadenylation and splicing before it becomes mature
mRNA.
Capping is the addition of methylated guanosine head on the 5´ end: The cap protects mRNA
from degradation and helps in nuclear export and ribosome binding.
Polyadenylation is the addition of poly (A) tail on the 3´ end: The poly-A tail protects mRNA
from degradation and aid in the export of mRNA from the nucleus; also aids in transcription
termination and in translation.
Splicing is the removal of introns and joining of exons together.

Importance of mRNA posttranscriptional modification

• Splicing: Helps in regulating gene expression because transcripts cannot leave the nucleus
to be translated until their introns are removed.
• The 5′ cap: Protects the nascent mRNA from degradation and assists in ribosome binding
during translation.
• The poly(A) tail: Protects the mRNA molecule from enzymatic degradation in the
cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and
translation
Figure 4.4 Messenger RNA post-transcription modification

4.3.4.2. Ribosomal RNA posttranscriptional modification

Ribosomal RNA (rRNA) is synthesised as precursor rRNA. Precursor rRNA undergoes
methylation and shortening to form mature rRNA. Mature rRNA combines with protein to
produce ribosome. A complete ribosome has two subunits. The prokaryotic ribosome is 70s (30s
+ 50s) while the eukaryotic ribosome is 80s (60s +40s).

Figure 4.5 Formation of prokaryotic (a) and eukaryotic (b) ribosomes.

4.3.4.3. Transfer RNA posttranscriptional modification
Precursor tRNA is converted into cloverleaf shaped mature tRNA which has the following
features: (i) D arm, (ii) T arm, (iii) anticodon and (iv) amino acid acceptor arm.

Figure 3.6 Mature tRNA

4.4 Transcription inhibiting drugs

Transcription inhibitors can be used as antibiotics against, for example, pathogenic bacteria
(antibacterial drugs) and fungi (antifungals). Rifampicin is an antibacterial drug which inhibits
prokaryotic DNA transcription while 8-Hydroxyquinoline is an antifungal drug which is a
transcription inhibitor.

4.5 REVIEW QUESTIONS

1. Explain differences between DNA replication and transcription.

2. Explain why the DNA molecule is referred to as the blue print for protein synthesis.
3. Explain the role of the promoter site during transcription.
4. Discuss posttranscriptional modification of RNA.
5. Differentiate between introns and exons and mention the type of RNA in which they
are found.
6. Describe the structure of each mature and functional prokaryotic RNA.
7. Explain the role of each RNA.

4.6 Summary
The synthesis of RNA using information from DNA is called transcription. The RNA molecule
is intermediate between DNA and proteins. The transcription process involves initiation,
elongation and termination. The stretch of DNA that is transcribed into an RNA molecule is
called a transcription unit. The section of DNA that holds the information for one polypeptide is
called a gene. Only one of the DNA strands is used as a template for the synthesis of RNA. This
is called the sense DNA strand. The other strand which is not transcribed is called the anti-sense
and it has the same code as the RNA transcript except for T in place of U.
TOPIC 5: TRANSLATION
5.1 Introduction
Translation, also called protein synthesis, is a process in which amino acids are assembled into
polypeptides based on the genetic information carried by mRNA derived from DNA. Translation
involves the participation of mRNA, tRNA and rRNA and a number of enzymes and other
proteins. The length of the polypeptide synthesised depends on the genetic information
(message) carried by the mRNA.

5.2 Objectives: At the end of this topic you should be able to:

1. To explain the role of mRNA and the genetic code

2. To explain the roles of tRNA and rRNA
3. To describe the mechanism of translation
4. To discuss posttranslational modification of newly synthesized polypeptides

5.3. Role of Messenger RNA

The mRNA carries the genetic code (triplet codon) derived from the template DNA. Messenger
RNA is a linear molecule which is a copy of the template DNA strand and varies in length
according to the length of the polypeptide that it codes for.
5.3.1 The genetic code
The genetic code is the whole sequence of nucleotides on the mRNA which is derived from the
DNA. The characteristics of the genetic code are; (i) It has codons in which the nucleotides of
mRNA are arranged as a linear sequence of successive triplets, (ii) it is non-overlapping meaning
that during translation, the codons do not overlap but are read sequentially, three at a time, (iii) it
is coma-less meaning that all the nucleotides are used to code for amino acids with none of them
used for punctuation, (v) it is universal meaning that it is the same for all organisms ranging from
virus to humans, (v) it has polarity meaning that it is always read in the 5’ → 3’ direction and
(vi) it is degenerate meaning that more than one codon may specify the same amino acid. Except
tryptophan and methionine which have a single codon each, all the other 18 amino acids have
more than one codon (vii) There are three codons that are not recognized by any tRNA: UAA,
UAG, and UGA. These are termed nonsense codons or stop codons, because they signal protein
synthesis to stop at that point.

5.3.2 Wobble hypothesis

The interaction between the codon in the mRNA and the anticodon in the tRNA needs to be
exact in first two nucleotide positions, but does not have to be so in the third position. Non-
standard base-pairing might occur between the third bases of the anticodon and of the codon. The
degenerate base (third base) is said to be in the wobble position.
1
Table 5.1 Table of the genetic code. Source: Jones and Karp. 1994. Introducing genetics. Page
200.

5.3.3 Role of Ribosomal RNA

Ribosomal RNA (rRNA) makes up nearly 80% of the total RNA of the cell. It combines with
protein to form organelles called ribosomes. Ribosomes are the site of protein synthesis. Each
ribosome has two subunits; a small and a large subunit. The function of the ribosome is to hold
in position the mRNA, tRNA and the associated enzymes involved in protein synthesis until a
peptide bond is formed between the adjacent amino acids. The ribosome has the A site
(aminoacyl site) and the P site (peptidyl site).

5.3.4 Role of transfer tRNA

Transfer RNA (tRNA) acts as an intermediate molecule between the triplet code of mRNA and
the amino acid sequence of the polypeptide chain. It is a small molecule with about 80
nucleotides. Each amino acid has its own tRNA which carries a triplet anticodon for the
particular amino acid. The anticodon is complementary to a specific codon on mRNA. The 3´
end of the tRNA carries the sequence 5´ -CCA-3´ which is used to attach a specific amino acid.

5.3.5 The mechanism of translation in prokaryotes

The main steps involved in translation may be summarised under the following headings: (i)
Binding of mRNA to ribosome (ii) amino acid activation (iii) attachment of amino acids to
specific tRNA (iv) polypeptide chain initiation (v) chain elongation (vi) chain termination (vii)
posttranslational modification.

2
5.3.5.1 Binding of mRNA to ribosome
The small (30S) ribosomal subunit binds to mRNA to form the 30S initiation complex. The
Shine Dalgarno (SD) sequence guides the mRNA to the binding site of the ribosome. This is
followed by the binding of the large (50S) subunit to the 30S initiation complex to form the 70S
initiation complex. Ribosomes are usually found in clusters linked together by one strand of
mRNA. This complex known as a polyribosome or polysome enables several copies of the same
polypeptide to be produced simultaneously.

Figure 5.1a. Binding of mRNA to prokaryotic ribosomal small (30S) subunit

Figure 5.1b. Binding of 50S to 30S initiation complex (A=Peptidyl site; A=aminoacyl site)

Figure 5.2. A polyribosome.

5.3.5.2Amino acid activation

Each target amino acid is activated by ATP using the enzyme aminoacyl-tRNA synthetase. There
is one enzyme for each amino acid.

3
Figure 5.3 Amino acid activation. Source: http://www.mdpi.com/ijms/ijms-15-
00401/article_deploy/html/images/ijms-15-00401f6-1024.png.

5.3.5.3 Attachment of activated amino acid to tRNA

The activated amino acid is then attached to its specific tRNA at the CCA end of the tRNA.
After this attachment, the tRNA complex is ready to transport the amino acid to the ribosome.

Figure 5.4. Attachment of amino acid to tRNA. Source: http://www.mdpi.com/ijms/ijms-15-

00401/article_deploy/html/images/ijms-15-00401f6-1024.png.

Figure 5.5a. Attachment of f-met to its tRNA

4
Figure 5.5b. Attachment of met to its tRNA
5.3.5.4 Polypeptide chain initiation

The tRNA with the anti-codon UAC for the codon AUG binds both amino acids - methionine
(met) and formyl methionine (f-met). Out of the two amino acids, f-met is the one carried to the
P site of the ribosome/mRNA complex to initiate the polypeptide chain. On the other hand, the
amino acid met is carried to the A site whenever the codon AUG reached the A site during the
process of protein synthesis. Special proteins called initiation factors are involved in chain
initiation.

Figure 5.6 Attachment of f-met-tRNA complex to the P site of the initiation complex.

5.3.5.4 Chain elongation

The second amino acid is brought to the A site by its tRNA. The ribosome then translocates to
the right by one codon, releasing the unloaded f-met. Meanwhile the second amino acid forms a
peptide bond with f-met by the action of the enzyme peptidyl transferase which is located in the
large subunit. The resulting dipeptide is carried by the second tRNA which is now located in the
P site.

5
Then the third amino acid is brought to the A site by its specific tRNA and it forms a peptide
bond with the dipeptide as the ribosome translocates to the right by one codon. The process is
repeated as more amino acids are added, one at a time, to the growing polypeptide chain as the
ribosome moves down the mRNA strand in the 5' → 3' direction of mRNA. Proteins called
elongation factors are involved in chain elongation.

Figure 5.7 Binding of the second amino acid to the A site of a ribosome. Source: Adapted
from Stansfield, 1991. Outline of Theory and problems of Genetics. Page 279.

Figure 5.8 Binding of the more amino acids to the growing peptide chain. Source: Adapted
from Stansfield, 1991. Outline of Theory and problems of Genetics. Page 279.

6
5.3.5.5 Chain termination
When one of the stop codons (UAA or UAG or UGA) reaches the A site, the synthesis of the
polypeptide chain stops. None of the tRNA molecules is able to bind to the stop codon but
instead special proteins called release factors bind to the stop codon. At this point the newly
made polypeptide chain is released and the ribosome is detached from the mRNA and split into
its subunits. The mRNA is broken down while the ribosomal subunits are recycled.
5.3.5.6 Post-translational modification
In order to achieve its biologically active form, the new polypeptide must fold into its proper
three-dimensional conformation. Before or after folding, the new polypeptide may undergo
enzymatic processing, including (i) removal of some amino acids; (ii) addition of functional
groups to certain amino acid; (iii) and attachment of oligosaccharides.
Insulin is an example of a polypeptide which undergoes post-translational modification. It is
synthesised as inactive pro-insulin and must be modified for it to become active insulin.

Figure 5.9 Formation of insulin from pro-insulin. Source: http://www.chemistrylearning.

com/humulin-synthetic-insulin.

5.4 REVISION QUESTIONS

1. Differentiate between the genetic code and a codon.

2. Define a gene.

3. Describe gene expression.

7
4. Explain the experiments carried out by Francis and his co-workers to confirm that the
genetic code is formed by triplet nucleotides.

5. Calculate the number of possible combinations that can arise from the triplet codon
system using the four nucleotides of RNA and comment on the significance of this
number.

6. Explain four features of the genetic code.

7. How many amino acids does each three-base triplet on the mRNA molecule code for?

8. State the start codon and mention the amino acid that it codes for.

9. What do the tree codons, UAA, UAG and UGA code for? Explain the role played by
these codons during protein synthesis.

10. State three differences between DNA replication and transcription.

11. Why is the DNA molecule referred to as the blue print for protein synthesis?

12. Discuss posttranscriptional modification.

13. What is the role of the promoter site during transcription?

14. Describe the structure of each mature prokaryotic RNA.

15. Explain the role of each prokaryotic RNA.

16. Differentiate between introns and exons and mention the type of RNA in which they
are found.

17. Which part of a ribosome contains a tRNA molecule that is attached to a growing
polypeptide chain?

18. During translation, an mRNA molecule has a codon with the triplet sequence 5´-
AUC-3´. (i) Write the anticodon sequence in a tRNA molecule that would pair with
this codon. (ii) Name the amino acid carried by this tRNA (iii) write the
corresponding sequence on the DNA from which the mRNA was transcribed..

19. What kind of bonds join amino acids in a polypeptide chain?

20. Discuss post translational modifications.

21. Differentiate the functions of the three types of RNA.

8
5.5 SUMMARY
Translation, also called protein synthesis, is a process in which amino acids are assembled into
polypeptides based on the genetic information carried by mRNA derived from DNA. Translation
involves the participation of mRNA, tRNA and rRNA and a number of enzymes and other
proteins. The length of the polypeptide synthesised depends on the genetic information
(message) carried by the mRNA. The main steps involved in translation may be summarised
under the following headings: (i) Binding of tRNA to ribosome (ii) amino acid activation (iii)
polypeptide chain initiation (iv) chain elongation (v) chain termination (vi) posttranslational
modification.

9
TOPIC 6 WATER

6.1 INTRODUCTION

• Water is the most abundant compound in living organisms, making up 60-95% fresh
mass of all living organisms.
• Water is important to living organisms because (i) it is an important chemical constituent
of living cells and (ii) it provides a habitat for aquatic organisms.
• Water has special physical and chemical properties because of its small size, its polarity
and due to hydrogen-bonding between its molecules.

6.2 DIPOLAR NATURE OF WATER

• Polarity is the unequal charge distribution over a molecule.

• In water one end of the molecule is slightly positive and the other slightly negative. This
is known as dipole.
• The electronegative oxygen atom (on one water molecule) attracts the electrons of the
hydrogen atoms (of surrounding water molecules); Water molecules therefore have an
electrostatic attraction for each other.
• Each oxygen atom has two partial negative charges while each hydrogen atom has one
partial positive charge. Hydrogen bonds are longer and weaker than covalent O-H bonds.

Figure 6.1 The dipolar nature of the water molecule is shown by (a) ball-and-stick and (b) space-filling models. In
(c) a hydrogen bond is formed between the oxygen of the upper and hydrogen of the lower molecule.

1
 • Because the two non-bonding pairs remain closer to the oxygen atom, these exert a
stronger repulsion against the two covalent bonding pairs, effectively pushing the two
hydrogen atoms closer together. The result is a distorted tetrahedral arrangement in which
the H—O—H angle is 104.5°.

6.3 HYDROGEN BOND FORMATION

• The attractions between hydrogen and oxygen atoms of different water molecules are
called hydrogen bonds.
• These weak bonds are also formed between (i) the oxygen atoms of water molecules and
hydrogen atoms of different molecules such as acids and (ii) the hydrogen atoms of water
and OH groups of other molecules such as alcohols.

6.4 IMPORTANCE OF WATER AS A SOLVENT

• Water is a good solvent for ionic substances like salts whose charged particles dissociate
in water and for some non-ionic substances like sugars and simple alcohols which contain
charged (polar) groups such as the hydroxyl (-OH) within the molecules.
• When substances dissolve in water they become more chemically reactive than when
they are in the solid form.
• Therefore, the majority of cellular reactions take place in aqueous solution.
• The solvent properties of water make it act as a transport medium, in blood, lymphatic
system, excretory system and the alimentary canal of animals as well as in xylem and
phloem tissues of plants.
2
Sodium ion, chloride ion , water Hydration of the sodium ion Hydration of the chloride ion by
molecule and sodium chloride by water molecules water molecules
crystal
Figure 6.2 The ionic charges of sodium chloride are partially neutralized and the electrostatic
attractions needed for crystal formation are weakened.

• Non-polar substances such as lipids are immiscible with water (insoluble) and are said to
be hydrophobic (water-hating).
• Hydrophobic interactions exist between different non-polar molecules which are mixed
with water and these are important in maintaining the stability of membranes, many
protein molecules, nucleic acids and other sub-cellular structures.

Figure 3.3 The formation of micelles by phospholipid molecules in water

6.5 ROLE RELATED TO WATER’S HIGH SPECIFIC HEAT CAPACITY

• The specific heat capacity of water is the amount of heat, in joules, that is required to
raise the temperature of 1 kg of water by 1°C.

3
 • Water has a high heat capacity, meaning that a large increase in heat energy results in a
relatively small rise in temperature.
• This is because much of the energy is used in weakening the hydrogen bonds between the
water molecules.
• This means that temperature fluctuations in water are minimised as a result of its high
heat capacity.
• Biochemical processes in living cells, therefore take place within a narrow temperature
range, at constant rates and are usually not affected by changes of temperature of the
environment.
• Water therefore provides a very constant external environment for many cells and
organisms.

6.6 ROLE RELATED TO WATER’S HIGH LATENT HEAT OF VAPORIZATION

• Latent heat of vaporisation is the amount of the heat energy required to vaporise a liquid.
Water has a high latent heat of vaporisation meaning that a large amount of heat can be
lost with minimal loss of water from the organism.
• This heat is used to overcome the cohesive (forces (hydrogen bonds) between water
molecules so that they can escape as a gas.
• As a result, water has an unusually high boiling point for such a small molecule.
• The energy absorbed by water molecules to evaporate results in the loss of energy from
their surroundings thus causing a cooling effect in the surroundings.
• Through sweating and panting mammals cool themselves while leaves are cooled down
during transpiration.

6.7 ROLE RELATED TO WATER’S DENSITY

• The density of water decreases at temperatures below 4°C and because of this ice tends to
float.
• Each water molecule, in ice, forms the maximum of four hydrogen bonds, creating a
regular lattice.

4
 • The crystal lattice of ice makes it less dense than liquid mater, thus ice floats on liquid
water.
• In liquid water, at room temperature and atmospheric pressure, each water molecule
forms an average of 3.4 bonds with other water molecules.
• Since ice floats, it forms at the surface of water first and at the bottom last.
• Therefore, ice insulates the water below it, increasing the chances of survival of
organisms in the water during the cold season in temperate climates.
• The fact that water below 4 °C tends to rise to the surface also helps to maintain
circulation in large bodies of water (lakes and oceans).
• This helps in bringing nutrients from the sediments (floor) of lakes and oceans to the
surface (nutrient cycling) and helps organisms (living things) to reach and stay at greater
depths.

Figure 6.4. Hydrogen bonding in ice.

6.8 ROLE RELATED TO WATER’S COHESION AND SURFACE TENSION

• Cohesion is the force which makes similar molecules stick together.
• At the surface of a liquid, such as water, a force called surface tension exists between the
molecules as a result of cohesion.
• This creates a ‘thin skin’ over the water surface. Water has a higher surface tension than
any other liquid.
• The high cohesion of water molecules helps in the movement of water in cells especially
in the translocation of water through the xylem in plants.
• In addition, many small aquatic organisms are able to float or skate on water because of
its high surface tension.

5
6.9 ROLE RELATED TO WATER’S ADHESION PROPERTIES

• The attraction between water molecules and other different substances is called adhesion.
• Water, through adhesion, makes things wet by sticking to their surfaces.
• Adhesion is important in the movement of water along the walls of vascular tissues in
plants and along blood vessels in mammals.

6.10 CAPILLARITY IN WATER

• This is the ability of water to flow upwards in narrow vessels without the assistance of
external forces like gravity or air pressure.
• Capillarity action is due to the action of cohesion and adhesion which together cause the
liquid to work against gravity and it occurs when forces of adhesion to the vessel walls
are stronger than the forces of adhesion between the liquid molecules.

6.11 REVISION EXERCISE

6.12 SUMMARY

Water has many chemical properties that make it very essential for life support. These include
the dipolar nature of the molecule and the hydrogen bonds that hold several water molecules
together. These properties make it an excellent solvent, a good biochemical reaction medium, a
buffer and a chemical that supports plant life in various ways including offering structural
support, as a cooling agent and as a reactant in photosynthesis

6
TOPIC 7 CARBOHYDRATES

7.1 INTRODUCTION

• Carbohydrates (hydrates of carbon) are compounds with the general formula Cx(H2O)y,
where x and y are variable numbers.
• The proportions of hydrogen and oxygen are 2:1 (same as in water)
• Carbohydrates are either aldehyde (aldoses) or ketone (ketoses) in nature and they all
contain several hydroxyl (OH) groups.
• Aldoses (with a terminal carbonyl group) are more easily oxidised than ketoses (with a
non-terminal carbonyl group)
• Hence aldoses act as reducing agents.
• Carbohydrates are divided into three main classes; mono-, di- and polysaccharides. Their
names end in a suffix -ose.

7.2 MONOSACCHARIDES

• Monosaccharides are the simplest form of carbohydrates made up of single sugar units
(monomers).
• They are used by cells as a direct source of energy.
• Monosaccharides are classified according to the number of carbon atoms they have.
Trioses have three carbons (3C), tetroses are 4C, pentoses are 5C, hexoses are 6C and
heptoses are 7C.
• Of these, trioses, pentoses and hexoses are the most common.
• Monosaccharides exist either in open chain forms or in ring forms.
• The open chain forms can exist as different isomers of a given monosaccharide.
• The most common isomer is the D-isomer (optical isomer) which rotates polarized light
to the right (dextro = D). The L-isomer is rare and rotates polarised light to the left (Levo
= L).

1
Figure 7.1. Structures of naturally occurring D-ribose (five carbons), D-glucose (six carbons)
and their synthetic isomers L-ribose (five-carbons) and L-glucose (six carbons).

7.2.1 TRIOSES
• There are two biologically important triose sugars i.e. (i) glyceraldehyde which has two
optical isomers - L-Glyceraldehyde and D-Glyceraldehyde and dihydroxyacetone, a
ketotriose with its carbonyl group located at the center the chain.

• During glycolysis in cellular respiration, fructose-1, 6-diphosphate is broken down into

glyceraldehyde-3-phosphate and dihydroxyacetone phosphate producing either lactic acid
or pyruvic acid and ATP.

Figure 7.2. Structures of the triose sugars glyceraldehyde and dihydroxyacetone

7.2.2 PENTOSE SUGARS

• Pentoses are 5- carbon sugars with the formula C5H10O5 e.g. ribulose, ribose, and
deoxyribose.
• Ribulose is an important CO2 acceptor during photosynthesis while ribose and
deoxyribose are important components of nucleic acids.

2
7.2.2.1 RIBOSE AND DEOXYRIBOSE

• These 5C sugars are part of the structure of nucleic acids (RNA and DNA).
• Ribose is a constituent of RNA while deoxyribose is a constituent of DNA.
• When carbon 2 has one OH group and one H atom, the result is ribose and when it has
two H atoms, the result is deoxyribose.

D-ribose open chain D-deoxyribose open chain

(aldose) (aldose)

Figure 7.3. Structures of D-ribose and D-deoxyribose (five-carbon sugars).

• Carbon 4 combines with carbon 1 of the open chain of both ribose and deoxyribose
resulting in the formation of a five membered furanose ring.
• The oxygen of carbon 4 links with carbon 1 to form a closed ring.
• The oxygen of the C=O bond on C1 combines with hydrogen of C4 to form an OH group
which is above the plane in β ribose and deoxyribose and below the plane in α ribose and
deoxyribose.
• The α and β forms are structural (stereo) isomers and are as a result of the asymmetric
(chiral) C1 which is attached to four different groups i.e. OH, H, O and C.

3
Figure 7.4. Formation of a ribose furanose ring. The ring which is at right-angles to the plane of
the paper can result into α-D-ribose (with OH group on C1 below the ring) or β-D-ribose (with
OH group on C1 above the ring).

Figure 7.5. Formation of a deoxyribose furanose ring. The ring which is at right-angles to the
plane of the paper can result into α-D-deoxyribose (with OH group on C1 below the ring) or β-
D-deoxyribose (with OH group on C1 above the ring).

7.2.3 HEXOSE SUGARS

• Hexoses are 6C sugars with the formula C6H12O6. Examples include glucose, fructose
and galactose.
• They are a direct source of energy and are oxidised during cellular respiration.
• Glucose is the most common respiratory monosaccharide.
• Hexoses are used in the synthesis of disaccharides (2 monomers), oligosaccharides (2-10
monomers) and polysaccharides (more than 10 monomers).

7.2.3.1 GLUCOSE

• Glucose (grape sugar) is a simple monosaccharide found in plants.

• It is an important product of photosynthesis and is a fuel for cellular respiration.
4
 • Glucose is soluble in water because its hydroxyl groups form hydrogen bonds with water
molecules.
• The open chain form of glucose is an aldehyde (aldose) whose carbon 1 combines with
the OH group of carbon 5 of the to give a six-membered pyranose ring.
• The ring structure results in carbon 1 atom becoming asymmetric or chiral (it forms four
single bonds with four different atoms and groups).
• When the OH group on carbon 1 is below the ring the result is the α-isomer of glucose
and when the OH group is above the ring the result is the β-isomer.
• The existence of α- and β-glucose leads to formation of a variety of the dimers and
polymers of glucose such as starch and glycogen (from α-glucose) and cellulose (from β -
glucose).
• Glucose is a powerful reducing agent because of the presence of the aldehyde group at
carbon 1 in the open chain (aldose).
• The commonly occurring glucose is the D-isomer while the synthetic L-isomer is
commonly used as a low-calorie sweetener and as a laxative.
• Living organisms cannot use L-glucose as an energy source because they do not have the
enzyme to break it down.

Figure 7.6 Formation of the glucose pyranose ring.

5
7.2.3.2 GALACTOSE

• Galactose is a water soluble monosaccharide (aldose sugar)

• It is a structural isomer of glucose.
• While on OH group on C4 of glucose is below the plane, the OH group on C4 of
galactose is above the plane
• It forms a pyranose ring which differs from glucose at C4 where the OH group is above
the plane in galactose and below the plane in glucose.
• Galactose combines with glucose to form a dimer called lactose (milk sugar) which is
present in milk.
• Galactose is a powerful reducing sugar because its open chain has an aldehyde group.

Figure 7.7. Formation of the galactose pyranose ring.

7.2.3.3 FRUCTOSE

• Fructose (fruit sugar) is a water-soluble monosaccharide found in honey, fruits, flowers,

berries, and in root crops.
• The open chain of fructose is a ketone (ketose) whose carbon 2 combines with the OH
group of carbon 5 to form a five-membered furanose ring.
• Like glucose, fructose is used as a source of energy in cellular respiration.
• Fructose is a weak reducing sugar because its open chain structure is able to isomerise

6
 into an aldose (glucose).
• Carbon 2 of fructose is asymmetric leading to the formation of the α- and β- isomers like
in glucose. Fructose is a structural isomer of glucose.

Figure 7.8. Formation of the fructose furanose ring.

7.2.3.3 HEXOSE ISOMERS

• Isomers are molecules that have the same molecular formula, but have a different
arrangement of the atoms in space.
• Examples of monosaccharide isomers include:
o (i) optical isomers: molecules which rotate polarised light to the right (D) or left
(L) e.g. D-glucose and L-glucose;
o (ii) stereoisomers isomers: molecules which have the same molecular formula
and structural formula but differ in the spatial arrangement of atoms in the
molecule e.g. (a) α-D glucose and β-D glucose and (b) and (b) glucose and
galactose;
o (iii) structural isomers: moecules which have the same molecular formula but
different structural formulae e.g glucose (aldose) and fructose (ketose)

7
7.3 DISACCHARIDES

• Disaccharides are made up of two sugar units (dimers). They are crystalline, sweet to
taste and readily dissolve in water.
• They are formed by the condensation (dehydration) reaction between two
monosaccharides which are joined together through a glycosidic bond.
• They are temporary energy stores which are broken down by hydrolysis into their
different monomers which are then used to produce energy through cellular respiration.
• The most common disaccharides are sucrose, lactose and maltose.

7.3.1 SUCROSE

• Sucrose (cane sugar) is composed of glucose and fructose which are linked through a 1,2
glycosidic bond between C1 of α-glucose and C2 of β-fructose.
• Its molecular formula of sucrose is C12H22O11.
• It is a temporary energy store which is broken down into glucose and fructose which are
then used in cellular respiration.
• It is most abundant in plants where it is translocated in large quantities through the
phloem tissue.

Figure 7.9. Formation and breakdown of sucrose

• Sucrose is a non reducing sugar because it is joined through carbon 1 of glucose and
carbon 2 of fructose that contain the aldehyde and keto groups which have the reducing
properties in the individual open chain structures.
• In industry, it is used as a sweetener, for baking, confectionery and as a food
preservative.

8
7.3.2 LACTOSE
• Lactose (milk sugar) is composed of galactose and glucose which are linked through a β–
(1, 4) glycosidic bond between C1 of β-galactose and C4 of either α-glucose or β-glucose.
• It has a formula of C12H22O11 and is water soluble
• The formation and breakdown of lactose is illustrated in Figure 4.10.

Figure 7.10. Formation and breakdown of lactose

• Lactose is a reducing sugar because carbon 1 of its glucose is free and can form an open
chain to expose the aldehyde which has the reducing properties.
• It can be broken down into galactose and glucose which are then used to generate energy
in tissue respiration.
• It can be extracted from sweet whey and is added to tablets as filler and to some types of
beer (stouts) to reduce the bitter taste.

7.3.3 MALTOSE

• Maltose (malt sugar) is water soluble and is composed of two glucose molecules which
are linked through an α 1→4 glycosidic linkage.
• It is a reducing sugar because carbon 1 on one of its glucose monomers is free and can
form an open chain to expose the aldehyde which has the reducing properties.

Figure 7.11. Formation and breakdown of maltose

9
 • Maltose is normally produced through the hydrolysis (digestion) of starch by the enzyme
amylase during animal digestion and in germinating seeds.
• Maltose is then broken down into glucose units by the enzyme maltase.
• It is an important intermediate during the production of fermented beverages (e.g. beers)
from cereals such as barley, millet, maize and rice.

7.4 POLYSACCHARIDES

• Polysaccharides are non-sweet, macromolecules which are insoluble or slightly soluble in

water.
• They are formed by the condensation (dehydration) reactions between many
monosaccharides (10 or more).
• Polysaccharides are used either as food and energy stores (e.g. starch and glycogen) or as
structural materials (e.g. cellulose and chitin).
• Starch and glycogen are suitable as energy storage molecules because they:
o are insoluble in water
o don’t affect the osmotic potential of the cell
o are highly branched and fold into compact shapes within limited space
o are easily converted to disaccharides and monosaccharides by hydrolysis
(digestion).

• Cellulose and chitin are suitable as structural materials because they:

o are insoluble in water
o are made of long thread (fibres)
o have very high tensile strength.

7.4.1 STARCH
• This is a polymer of α-glucose monomers which first join to form maltose.
• The maltose units then join to form starch.

10
• It is insoluble in cold water because it is a very long molecule with many glycosidic
bonds between glucose monomers and it’s a tightly packed structure.
• Starch has two components:
o Amylose which has a straight chain structure, consisting of thousands of α-
glucose molecules linked through α-1,4 glycosidic bonds and coils into a helix
forming a compact shape.
▪ A suspension of amylose in water gives a blue-black colour with iodine
solution because the iodide complex slips into the α-helix coil.
▪ It constitutes 20-30% of starch.
o Amylopectin which has a similar structure to amylose but has additional branches,
formed by α-1, 6 glycosidic bonds and its chains are shorter.
▪ A suspension of amylopectin in water gives a red-violet colour with iodine
solution because there is little interaction between the iodide complex and
the amylopectin.
▪ It constitutes 70-80% of starch.
• Starch which is a major energy store in plants, is the most common carbohydrate in
human staple foods such as rice, wheat, maize, cassava and potatoes.
• Starch is easily broken down into maltose (disaccharide) and glucose (monosaccharide)
by hydrolysis (digestion).
• In industry, starch is used as a thickening agent, stabilizer or as a prebiotic.

11
Figure 7.12. Synthesis and breakdown of amylose (straight chain starch)

Figure 7.13. Synthesis and breakdown of amylopectin (branched starch)

12
7.4.2 GLYCOGEN

• Glycogen is the animal equivalent of starch (amylopectin) and is made from α-glucose.
• It is water insoluble because of the numerous α(1-4) and α(1-6) bonds between its
glucose monomers and its compact structure.
• It exists in form of granules in the cytoplasm of many types of cells but is stored mainly
in the liver and muscle cells of vertebrates.
• Glycogen is hydrolysed into glucose for use in respiration when need arises.
• Although it is similar to amylopectin, its branches are shorter and more frequent.
• The glucose chains of glycogen are organized in branches of a tree originating from a
protein molecule at the centre of the structure.

Figure 7.14. Structure of glycogen

13
7.4.3 CELLULOSE

• Cellulose is a water insoluble polymer of β-glucose with -CH2OH groups alternating

above and below the plane of the long unbranched cellulose molecule.
• The hydroxyl (OH) groups which project outwards from each chain form hydrogen bonds
with neighbouring chains.
• Neighbouring cellulose molecules lie close together in layers and form rigid structures
called microfibrils, which are arranged in larger bundles called macrofibrils.
• These bundles are arranged in layers and have tremendous tensile strength.
• The cellulose layers are fully permeable to water and solutes which are important in the
functioning of plant cells.
• Cellulose is the most abundant organic compound on earth and is the major structural
material of plant cell walls.

(a) (b)

Figure 7.15. Structures of cellulose (a), a fibril and a microfibril (b)

• Cattle, horses and other ruminant mammals as wells as termites have bacteria living in
their digestive systems that are able to digest cellulose into soluble sugars which are then
used by both the bacteria and their hosts (a relationship called symbiosis).
• Cellulose is important in human diets as dietary fibre. In industry, cellulose is used to
produce paper and cotton thread. There is some research aimed at converting cellulose
into biofuels.

14
7.4.4 CHITIN

• Chitin is an unbranched polymer of N-Acetyl-D-glucosamine. It is found in cell walls of

fungi and in exoskeletons of insects, crabs, and shrimps.
• It is similar to cellulose, but the hydroxyl group of carbon 2 of each glucose unit has been
replaced with an acetamido (NH(C=O)CH3) group.

Figure 7.16. Structure of chitin

7.5 METABOLISM OF CARBOHYDRATES

Table 4.1 The breakdown of various carbohydrates

# Carbohydrate Enzyme (s) Products
1 Starch Amylase Maltose
2. Maltose Maltase Glucose
2. Sucrose Sucrase/Invertase Glucose + Fructose
3. Lactose Lactase Galactose + Glucose
4. Glycogen Glycogen phosphorylase Glucose
5. Glucose Various glycolytic enzymes CO2 + H2O + Energy
6. Fructose Various glycolytic enzymes CO2 + H2O + Energy
7. Galactose Various glycolytic enzymes CO2 + H2O + Energy

15
 AMINO ACIDS
LESSON II
BIO 1401
Dr Sydney Malama

Introduction

An amino acid is any of a group of organic molecules that consist of a basic amino
group (―NH2), an acidic carboxyl group (―COOH), and an organic R group (or side
chain) that is unique to each amino acid. The term amino acid is short for α-amino
[alpha-amino] carboxylic acid. Each molecule contains a central carbon (C) atom,
called the α-carbon, to which both an amino and a carboxyl group are attached. The
remaining two bonds of the α-carbon atom are generally satisfied by a hydrogen (H)
atom and the R group. The formula of a general amino acid is:

Acid-base properties

Another important feature of free amino acids is the existence of both a basic and an
acidic group at the α-carbon. Compounds such as amino acids that can act as either an
acid or a base are called amphoteric. The basic amino group typically has a pKa between
9 and 10, while the acidic α-carboxyl group has a pKa that is usually close to 2 (a very
low value for carboxyls). The pKa of a group is the pH value at which the concentration
of the protonated group equals that of the unprotonated group. Thus, at physiological
pH (about 7–7.4), the free amino acids exist largely as dipolar ions or “zwitterions”
(German for “hybrid ions”; a zwitterion carries an equal number of positively and
negatively charged groups). Any free amino acid and likewise any protein will, at some
specific pH, exist in the form of a zwitterion. That is, all amino acids and all proteins,
when subjected to changes in pH, pass through a state at which there is an equal number
of positive and negative charges on the molecule. The pH at which this occurs is known
as the isoelectric point (or isoelectric pH) and is denoted as pI. When dissolved in water,
all amino acids and all proteins are present predominantly in their isoelectric form.
Stated another way, there is a pH (the isoelectric point) at which the molecule has a net
zero charge (equal number of positive and negative charges), but there is no pH at which
the molecule has an absolute zero charge (complete absence of positive and negative
charges). That is, amino acids and proteins are always in the form of ions; they always
carry charged groups. This fact is vitally important in considering further the
biochemistry of amino acids and proteins.
Group I: Nonpolar amino acids
Group I amino acids are glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, and tryptophan. The R groups of these amino acids have
either aliphatic or aromatic groups. This makes them hydrophobic (“water fearing”). In
aqueous solutions, globular proteins will fold into a three-dimensional shape to bury
these hydrophobic side chains in the protein interior. The chemical structures of Group
I amino acids are:
Isoleucine is an isomer of leucine, and it contains two chiral carbon atoms. Proline is
unique among the standard amino acids in that it does not have both free α-amino and
free α-carboxyl groups. Instead, its side chain forms a cyclic structure as the nitrogen
atom of proline is linked to two carbon atoms. (Strictly speaking, this means that proline
is not an amino acid but rather an α-imino acid.) Phenylalanine, as the name implies,
consists of a phenyl group attached to alanine. Methionine is one of the two amino acids
that possess a sulfur atom. Methionine plays a central role in protein biosynthesis
(translation) as it is almost always the initiating amino acid. Methionine also provides
methyl groups for metabolism. Tryptophan contains an indole ring attached to the alanyl
side chain.

Group II: Polar, uncharged amino acids

Group II amino acids are serine, cysteine, threonine, tyrosine, asparagine, and
glutamine. The side chains in this group possess a spectrum of functional groups.
However, most have at least one atom (nitrogen, oxygen, or sulfur) with electron pairs
available for hydrogen bonding to water and other molecules. The chemical structures
of Group II amino acids are:
Group III: Acidic amino acids

The two amino acids in this group are aspartic acid and glutamic acid. Each has a
carboxylic acid on its side chain that gives it acidic (proton-donating) properties. In an
aqueous solution at physiological pH, all three functional groups on these amino acids
will ionize, thus giving an overall charge of −1. In the ionic forms, the amino acids are
called aspartate and glutamate. The chemical structures of Group III amino acids are

The side chains of aspartate and glutamate can form ionic bonds (“salt bridges”), and
they can also function as hydrogen bond acceptors. Many proteins that bind metal ions
(“metalloproteins”) for structural or functional purposes possess metal-binding sites
containing aspartate or glutamate side chains or both. Free glutamate and glutamine
play a central role in amino acid metabolism. Glutamate is the most abundant excitatory
neurotransmitter in the central nervous system.
Group IV: Basic amino acids

The three amino acids in this group are arginine, histidine, and lysine. Each side chain
is basic (i.e., can accept a proton). Lysine and arginine both exist with an overall charge
of +1 at physiological pH. The guanidino group in arginine’s side chain is the most
basic of all R groups (a fact reflected in its pKa value of 12.5). As mentioned above for
aspartate and glutamate, the side chains of arginine and lysine also form ionic bonds.
The chemical structures of Group IV amino acids are

The imidazole side chain of histidine allows it to function in both acid and base catalysis
near physiological pH values. None of the other standard amino acids possesses this
important chemical property. Therefore, histidine is an amino acid that most often
makes up the active sites of protein enzymes.

The majority of amino acids in Groups II, III, and IV are hydrophilic (“water loving”).
As a result, they are often found clustered on the surface of globular proteins in aqueous
solutions.
Amino acid reactions

Amino acids via their various chemical functionalities (carboxyls, amino, and R groups)
can undergo numerous chemical reactions. However, two reactions (peptide bond and
cysteine oxidation) are of particular importance because of their effect on protein
structure.

PEPTIDE BOND

Amino acids can be linked by a condensation reaction in which an ―OH is lost from
the carboxyl group of one amino acid along with a hydrogen from the amino group of
a second, forming a molecule of water and leaving the two amino acids linked via an
amide—called, in this case, a peptide bond. At the turn of the 20th century, German
chemist Emil Fischer first proposed this linking together of amino acids. Note that when
individual amino acids are combined to form proteins, their carboxyl and amino groups
are no longer able to act as acids or bases, since they have reacted to form the peptide
bond. Therefore, the acid-base properties of proteins are dependent upon the overall
ionization characteristics of the individual R groups of the component amino acids.

Amino acids joined by a series of peptide bonds are said to constitute a peptide. After
they are incorporated into a peptide, the individual amino acids are referred to as amino
acid residues. Small polymers of amino acids (fewer than 50) are called oligopeptides,
while larger ones (more than 50) are referred to as polypeptides. Hence, a protein
molecule is a polypeptide chain composed of many amino acid residues, with each
residue joined to the next by a peptide bond. The lengths for different proteins range
from a few dozen to thousands of amino acids, and each protein contains different
relative proportions of the 20 standard amino acids.
TOPIC 8 LIPIDS

8.1 INTRODUCTION

• Lipids are a group of naturally occurring esters of fatty acids and alcohols.
• They include fats, waxes, fat-soluble vitamins (such as vitamins A, D, E, and K),
glycerides, phospholipids, and steroids (special type of lipids with a ring structure).
• They are water-insoluble and can be extracted from cells using organic solvents such
as ether, chloroform and benzene.
• The main biological functions of lipids include (i) energy storage (ii) cell signalling
(iii) structural support of cell membranes (iv) heat and electrical insulation (v)
buoyancy (vi) water proofing (vii) production of metabolic water and (viii) protection
of internal organs.

8.2 FATTY ACIDS

• A fatty acid is made of a hydrocarbon chain that terminates with a carboxylic acid
group [CH3(CH2)xCOOH].
• This makes the fatty acid molecule amphipathic meaning that it has a polar,
(hydrophilic) head and a nonpolar (hydrophobic) tail.

8.2.1 SATURATED FATTY ACIDS

• Fatty acids without any carbon-carbon double bonds are said to be saturated meaning
each carbon atom has the maximum number of hydrogen atoms.
• They have only carbon-carbon single bonds (C-C).
• The absence of double bonds makes saturated fatty acids rigid (not flexible).
• This rigidity makes fatty acids waxy solids at room temperature.
• An example of a saturated fatty acid is stearic acid which has 18 carbons, with a
chemical formula CH3(CH2)16COOH.
• Stearic acid is found in butter, cheese, coconut oil, meat, margarine and fried foods.
• It is mainly used in the production of detergents, soaps, shampoos and shaving
creams.

1
Figure 8.1. The structure of stearic acid an unsaturated fatty acid

8.2.2 UNSATURATED FATTY ACIDS

• Fatty acids with one or more carbon-carbon double bonds (C=C) are said to be
unsaturated meaning that some of the carbon atoms have less than the maximum
number of hydrogen atoms.
• Monounsaturated fatty acids have one carbon-carbon double bond while
polyunsaturated fatty acids have two or more.
• The presence of a double bonds creates a bend (kink) and this makes unsaturated fatty
acids flexible (fluid) and able to melt at much lower temperature than saturated fatty
acids.
• Oleic acid is an example of a mono unsaturated 18 carbon molecule with a chemical
formula CH3(CH2)7CH=CH(CH2)7COOH.
• It is an oily liquid at room temperature, found in olive oil, avocado, soya bean oil,
canola oil, sunflower oil and walnuts.

Figure 8.2. Structure of oleic acid a saturated fatty acid

2
8.2.2.1 CIS AND TRANS UNSATURATED FATTY ACIDS

• Cis unsaturated fatty acids have hydrogen bonds on the same side of the double bond
making them relatively more flexible than trans unsaturated fatty acids which have
hydrogen atoms on opposite sides of the double bond.

Figure 8.3. Structure of a cis and a trans carbon-carbon double bond.

8.2.3 GLYCEROL

• Glycerol (or glycerine) is a simple three-carbon poly alcohol which combines with fatty acids
in the synthesis glyceride lipids.
• It is colourless, viscous and sweet-tasting.
• The three hydroxyl groups make it water soluble and adsorbent (hygroscopic).
• Its chemical formula is C3H8O3.

Figure 8.4. Structure of glycerol (propane 1,2,3 triol or 1,2,3 propanetriol).

• It is produced from the lipids through saponification (reaction between sodium and a
glycerides).
• It is used as a solvent, sweetner, preservative or as an additive in food, pharmaceutical and
cosmetic products.

3
8.2.4 GLYCERIDES

• Glycerides are ester compounds of glycerol and fatty acids.

• Each of the three hydroxyl (-COH) groups each of glycerol can react with a carboxyl
group (-COOH) of a fatty acid to form an ester (-COOC-) bond.
• This is a condensation reaction and water is released in the process.
• A monoglyceride has one fatty acid chain; a diglyceride has two while a triglyceride
has three.

Figure 8.4. Formation of a triglyceride (lipid) from three fatty acids and glycerol by a
condensation reaction

• The long hydrocarbon chains of the fatty acids form hydrophobic tails which make
lipids insoluble in water.
• Triglycerides are the commonest lipids in nature and are classified as fats (if they are
solid at 20°C) and as oils (if they are liquid at 20°C).

8.2.5 PHOSPHOLIPIDS
• A phospholipids is a modified triglyceride in which one fatty acid has been replaced
by a phosphate group linked to a nitrogen-containing molecule.
• One of the two fatty acid chains of a phospholipid is saturated while the other chain is
unsaturated and has a bend or kink.
• Phospholipids form ball-shaped micelles or liposomes in water because of the
hydrophobic interactions between the fatty acid chains (tails) which tend to cluster
inwards away from water while the phosphate groups (heads) stick out into the
surrounding water.

4
 (a) (b)
Figure 8.6. Formation of a micelle (a) and a liposome (b) in water by phospholipids

• Phospholipids are found in the cell membrane where they form a phospholipid bilayer
which is important for the function of the cell membrane (Solomon and Berg, 1995
pages 70-71).

Figure 8.7. Structure of a phospholipid and the cell membrane bilayer

8.3.6 WAXES
• Waxes are mainly esters of fatty acids with long-chain alcohols.
• They are used as waterproofing material by plants and animals.
• In plants they form an additional protective layer on the cuticle of the epidermis of
leaves, fruits and seeds.

5
 • In vertebrate animals they are found in skin, fur and feathers while in insects they are
part of the exoskeleton (chitin).

Figure 8.5. Formation of a wax ester from a fatty acid and an alcohol.

• Beeswax is a special type of wax found in the honeycombs.

• The bees use it to store honey and to protect the larvae and pupae.
• It is used as a glazing agent for fruits, a coating agent in cheese and chewing gum, in
lip balm and lip stick, etc

• Carnauba wax also called Brazil wax or palm wax is a wax of the leaves of the
palm tree. It is used in automobile waxes, shoe polishes, instrument polishes,
floor and furniture waxes and polishes, dental floss and in food products such
as sweets,

8.3.7 STEROIDS

• These are special lipid compounds with four rings which are found in plants, animals
and fungi.

Steroid Cholesterol
Figure 8.8. Chemical structure of a steroid and of cholesterol

6
 • The large number of CH groups makes steroids non-polar.
• The most common type of animal steroid is cholesterol which is an important part of
the animal cell membrane structure and function.
• Cholesterol is also a precursor to fat-soluble vitamins and steroid hormones ((e.g. sex
hormones (Solomon and Berg, 1995 pages 70-71))

Figure 8.9. The interaction between cholesterol and phospholipids in the cell membrane.

7
 PROTEIN STRUCTURE
LESSON III

BIO1401

Dr Sydney Malama

Introduction

Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule.

Proteins are polymers specifically polypeptides formed from sequences of amino acids, the
monomers of the polymer. A single amino acid monomer may also be called a residue indicating
a repeating unit of a polymer. Proteins form by amino acids undergoing condensation reactions, in
which the amino acids lose one water molecule per reaction in order to attach to one another with
a peptide bond. By convention, a chain under 30 amino acids is often identified as a peptide, rather
than a protein. To be able to perform their biological function, proteins fold into one or more
specific spatial conformations driven by a number of non-covalent interactions such as hydrogen
bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the
functions of proteins at a molecular level, it is often necessary to determine their three-dimensional
structure. This is the topic of the scientific field of structural biology, which employs techniques
such as X-ray crystallography, NMR spectroscopy, and dual polarisation interferometry to
determine the structure of proteins.

Protein structures range in size from tens to several thousand amino acids. By physical size,
proteins are classified as nanoparticles, between 1–100 nm. Very large aggregates can be formed
from protein subunits. For example, many thousands of actin molecules assemble into a
microfilament.
 .

Interactive diagram of protein structure

A protein generally undergoes reversible structural changes in performing its biological function.
The alternative structures of the same protein are referred to as different conformational isomers,
or simply, conformations, and transitions between them are called conformational changes.

Levels of protein structure

There are four distinct levels of protein structure.

Primary structure

The primary structure of a protein refers to the sequence of amino acids in the polypeptide chain.
The primary structure is held together by peptide bonds that are made during the process of protein
biosynthesis. The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-
terminus) and the amino terminus (N-terminus) based on the nature of the free group on each
extremity. Counting of residues always starts at the N-terminal end (NH2-group), which is the end
where the amino group is not involved in a peptide bond. The primary structure of a protein is
determined by the gene corresponding to the protein. A specific sequence of nucleotides in DNA
is transcribed into mRNA, which is read by the ribosome in a process called translation. The
sequence of amino acids in insulin was discovered by Frederick Sanger, establishing that proteins
have defining amino acid sequences. The sequence of a protein is unique to that protein, and
defines the structure and function of the protein. The sequence of a protein can be determined by
methods such as Edman degradation or tandem mass spectrometry. Often, however, it is read
directly from the sequence of the gene using the genetic code. It is strictly recommended to use
the words "amino acid residues" when discussing proteins because when a peptide bond is formed,
a water molecule is lost, and therefore proteins are made up of amino acid residues. Post-
translational modification such as phosphorylations and glycosylations are usually also considered
a part of the primary structure, and cannot be read from the gene. For example, insulin is composed
of 51 amino acids in 2 chains. One chain has 31 amino acids, and the other has 20 amino acids.

Secondary structure

An α-helix with hydrogen bonds (yellow dots)

Secondary structure refers to highly regular local sub-structures on the actual polypeptide
backbone chain. Two main types of secondary structure, the α-helix and the β-strand or β-sheets,
were suggested in 1951 by Linus Pauling et al. These secondary structures are defined by patterns
of hydrogen bonds between the main-chain peptide groups. They have a regular geometry, being
constrained to specific values of the dihedral angles ψ and φ on the Ramachandran plot. Both the
α-helix and the β-sheet represent a way of saturating all the hydrogen bond donors and acceptors
in the peptide backbone. Some parts of the protein are ordered but do not form any regular
structures. They should not be confused with random coil, an unfolded polypeptide chain lacking
any fixed three-dimensional structure. Several sequential secondary structures may form a
"supersecondary unit".

Tertiary structure

Tertiary structure refers to the three-dimensional structure of monomeric and multimeric protein
molecules. The α-helixes and β-pleated-sheets are folded into a compact globular structure. The
folding is driven by the non-specific hydrophobic interactions, the burial of hydrophobic residues
from water, but the structure is stable only when the parts of a protein domain are locked into place
by specific tertiary interactions, such as salt bridges, hydrogen bonds, and the tight packing of side
chains and disulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the
cytosol (intracellular fluid) is generally a reducing environment.

Quaternary structure

Quaternary structure is the three-dimensional structure consisting of the aggregation of two or

more individual polypeptide chains (subunits) that operate as a single functional unit (multimer).
The resulting multimer is stabilized by the same non-covalent interactions and disulfide bonds as
in tertiary structure. There are many possible quaternary structure organisations. Complexes of
two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be
called a dimer if it contains two subunits, a trimer if it contains three subunits, a tetramer if it
contains four subunits, and a pentamer if it contains five subunits. The subunits are frequently
related to one another by symmetry operations, such as a 2-fold axis in a dimer. Multimers made
up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those
made up of different subunits are referred to with a prefix of "hetero-", for example, a
heterotetramer, such as the two alpha and two beta chains of hemoglobin.
Domains, motifs, and folds in protein structure

Protein domains. The two shown protein structures share a common domain (maroon), the PH
domain, which is involved in phosphatidylinositol (3,4,5)-trisphosphate binding

Proteins are frequently described as consisting of several structural units. These units include
domains, motifs, and folds. Despite the fact that there are about 100,000 different proteins
expressed in eukaryotic systems, there are many fewer different domains, structural motifs and
folds.

Structural domain

A structural domain is an element of the protein's overall structure that is self-stabilizing and often
folds independently of the rest of the protein chain. Many domains are not unique to the protein
products of one gene or one gene family but instead appear in a variety of proteins. Domains often
are named and singled out because they figure prominently in the biological function of the protein
they belong to; for example, the "calcium-binding domain of calmodulin". Because they are
independently stable, domains can be "swapped" by genetic engineering between one protein and
another to make chimera proteins.

Structural and sequence motif

The structural and sequence motifs refer to short segments of protein three-dimensional structure
or amino acid sequence that were found in a large number of different proteins.

Super secondary structure

The super secondary structure refers to a specific combination of secondary structure elements,
such as β-α-β units or a helix-turn-helix motif. Some of them may be also referred to as structural
motifs.

Protein fold

A protein fold refers to the general protein architecture, like a helix bundle, β-barrel, Rossman fold
or different "folds" provided in the Structural Classification of Proteins database. A related concept
is protein topology that refers to the arrangement of contacts within the protein.

Super domain

A super domain consists of two or more nominally unrelated structural domains that are inherited
as a single unit and occur in different proteins. An example is provided by the protein tyrosine
phosphatase domain and C2 domain pair in PTEN, several tensin proteins, auxilin and proteins in
plants and fungi. The PTP-C2 super domain evidently came into existence prior to the divergence
of fungi, plants and animals is therefore likely to be about 1.5 billion years’ old

Protein dynamics

A ribosome is a biological machine that utilizes protein dynamics on nanoscales

Proteins are not however strictly static objects, but rather populate ensembles of conformational
states. Transitions between these states typically occur on nanoscales, and have been linked to
functionally relevant phenomena such as allosteric signaling and enzyme catalysis. Protein
dynamics and conformational changes allow proteins to function as nanoscale biological machines
within cells, often in the form of multi-protein complexes. Examples include motor proteins, such
as myosin, which is responsible for muscle contraction, kinesin, which moves cargo inside cells
away from the nucleus along microtubules, and dynein, which moves cargo inside cells towards
the nucleus and produces the axonemal beating of motile cilia and flagella. "[I]n effect, the [motile
cilium] is a nanomachine composed of perhaps over 600 proteins in molecular complexes, many
of which also function independently as nanomachines...Flexible linkers allow the mobile protein
domains connected by them to recruit their binding partners and induce long-range allostery via
protein domain dynamics. "

Protein folding

As it is translated, polypeptides exit the ribosome as a random coil and folds into its native state.
Since the fold is determined by a network of interactions between amino acids in the polypeptide,
the final structure of the protein chain is determined by its amino acid sequence (Anfinsen's
dogma).

Protein stability

Protein stability depends upon a few factors such as 1) Non-covalent electrostatic interactions 2)
Hydrophobic interactions These interaction energies are from the order of 20-40 kJ/mol. Proteins
are very sensitive to changing temperatures and a change in temperature may result in unfolding
or denaturation. Protein denaturation may result in loss of function, and loss of native state.

X-ray crystallography and calorimetry indicates that there is no general mechanism that describes
the effect of temperature change on the functions and structure of proteins. This is due to the fact
that proteins do not represent a uniform class of chemical entities from an energetic point of view.
The structure and stability of an individual protein depends on the ratio of its polar and non-polar
residues. They contribute to the conformational and the net enthalpies of local and non-local
interactions.

Taking the weak intermolecular interactions responsible for structural integrity into consideration,
it is hard to predict the effects of temperature because there are too many unknown factors
contributing to the hypothetical free energy balance and its temperature dependence. Internal salt
linkages produce thermal stability, and whether cold temperature results in the destabilization of
these linkages is unknown.

In principle, the free energy of stabilization of soluble globular proteins does not exceed 50-100
kJ/mol. The stabilization is based on the equivalent of few hydrogen bonds, ion pairs, or
hydrophobic interactions, even though numerous intramolecular interactions results in
stabilization. Taking into consideration the large number of hydrogen bonds that take place for the
stabilization of secondary structures, and the stabilization of the inner core through hydrophobic
interactions, the free energy of stabilization emerges as small difference between large numbers.
Therefore, the structure of a native protein is not optimized for the maximum stability.
NUCLEIC ACIDS

Prepared by Dr. P. Khondowe

Introduction
• Nucleic acids are macromolecules through
which inheritance of character from parents to
offspring occurs.
• They are composed of carbon, hydrogen,
oxygen, nitrogen, and phosphorus.
• Cells store their information needed to control
their activities through nucleic acids.
• There are two types of nucleic acids,
deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA).
• These are structurally similar but perform
different functions within the cell.
• DNA is the genetic blueprint for building cells.
• RNA reads the information encoded by the DNA
and interprets it to make proteins.
Structure of Nucleic Acids
• Nucleic acids are polymers made of monomers
called nucleotides (or mononucleotides).
• Thus both RNA and DNA are polymers of
nucleotides.
• Nucleotides are in turn joined together to form
long nucleic acid molecules called
polynucleotides.
Structure of Nucleotides
• All nucleotides are composed of a nitrogenous
base attached to a sugar linked to a phosphate.
• Both RNA and DNA are synthesized from
combinations of four types of nucleotides that
differ in the nature of their nitrogenous bases.
• Therefore a mononucleotide is made of three
parts:
• (i) a sugar
• (ii) a phosphate a phosphate (—PO4-) group and
• (iii) a nitrogenous base.
• Sugar
• This is made of 5 carbons (hence a pentose).
• RNA and DNA differ in the type of sugar in the
nucleotide: ribonucloetides contain ribose
whereas deoxyribonucleotides possess
deoxyribose.
• So in DNA the sugar is deoxyribose, and in
RNA the sugar is ribose.
5’ CH 2OH OH 5’CH
2OH OH
O O
4’ 1’ 4’ 1’

3’ 2’ 3’ 2’

OH OH OH

Deoxyribose Ribose
• Nitrogenous bases
• These are ring compounds containing nitrogen
(hence, nitrogenous base).
• Nitrogenous bases have the characters of bases
because of the nitrogen they contain.
• DNA contains combinations of 4 different bases
with equal numbers of purines and pyrimidines.
• Nucleotides have five types of nitrogenous
bases.
• Two of these are large, double-ring molecules
called purines that are each found in both DNA
and RNA; the two purines are adenine (A) and
guanine (G).
• The other three bases are single-ring molecules
called pyrimidines that include cytosine (C, in
both DNA and RNA), thymine (T, in DNA only),
and uracil (U, in RNA only).
• Nucleoside:
• A structure containing only the sugar and the
base is known as a nucleoside.
• Where the sugar is deoxyribose the nucleotide
is called a deoxyribonucleotide.
• Where the sugar is ribose the nucleotide is
called a ribonucleotide.
• Thus the ribonucleotides are ATP, UTP, CTP,
and GTP.
• The deoxyribonucleotides are dATP, dTTP,
dCTP, and dGTP.
• The three parts of a mononucleotide are
joined by two condensation reactions:
• (a) one between the phosphate and the
sugar,
• (b) one between the sugar and the
nitrogenous base.
Synthesis of Polynucleotides
• Nucleic acids form from long polymers of nucleotides
linked by phosphodiester bonds that form between the
phosphate of one nucleotide and the sugar of the
adjacent nucleotide (by dehydration synthesis).
• The end of the polymer that terminates with a phosphate
group is deemed the 5-prime end (5′); the other end
terminates with a sugar and is the 3′ end.
• The nucleic acid has a polarity, conferred by its 5′ (“five-
prime,” —PO4 -) & 3′ (“three-prime,” —OH) ends, taken
from the carbon numbering of the sugar.
 Sugar

Phosphate Bases stick out

Sugar and phosphate

join here by a phosphodiester
bond formed by a
condensation reaction
Thymine

A DNA polynucleotide chain

showing adenine, thymine,
guanine, cytosine and
guanine
RNA Structure and Functions
• RNA is similar to DNA, but with two major chemical
differences.
• First, RNA molecules contain ribose sugars, in which the
C-2 is bonded to a hydroxyl group. (In DNA, a hydrogen
atom replaces this hydroxyl group.)
• Second, RNA molecules use uracil in place of thymine.
Uracil has a similar structure to thymine, except that one
of its carbons lacks a methyl (—CH3) group.
• Cells produce three main forms of RNA: transfer RNA
(tRNA), ribosomal RNA (rRNA), and messenger RNA
(mRNA).
• RNA molecules form single strands. These can fold into
complex structures and shapes or remain as long
thread-like molecules.
• RNA is produced by transcription (copying) from DNA,
and is usually single-stranded.
• The role of RNA in cells is quite varied: it carries
information in the form of mRNA, it is part of the
ribosome, in the form of ribosomal RNA (rRNA), and it
carries amino acids in the form of transfer RNA (tRNA).
• Transcription
• Transcription is the synthesis of mRNA using
DNA as the template.
• It takes place in the nucleus.
• mRNA forms through a reaction catalyzed by
RNA polymerase.
• The mRNA moves out of the nucleus to the
cytoplasm onto the surface of the ribosomes.
This is where protein synthesis occurs.
• mRNA is itself formed as a complementary
strand to DNA. It is like a mirror image of DNA.
• tRNA and translation
• The process of translation to make proteins from
mRNA requires transfer RNA (tRNA).
• tRNA is attached to amino acids through a
chemical reaction.
• It then transfers the attached amino acid to the
place where the protein chain is growing.
 DNA Structure and Functions

• A DNA molecule is made up of 2 polynucleotide

strands twisted around each other.
• The sugars and phosphates form the backbone
of the molecule.
• The bases point inward from the backbone.
 Base Pairing in DNA

• The two strands of DNA are lined up with the bases

pointing towards each other.
• The strands run in opposite directions i.e they are
anti-parallel with one strand running 5' to 3' while the
other runs 3' to 5'
• The shape and chemical nature of the bases mean
that (normally) they only pair up in particular ways
specifically:
• Guanine (a purine) pairs with Cytosine (a pyrimidine)
(GC)
• Adenine ( the other purine) pairs with Thymine (the
other pyrimidine)
The structure of DNA
was discovered by an
American scientist
(James Watson) and a
British scientist (Francis
Crick) based on the
work of Rosalind
Franklin and Maurice
Wilkins.
In 1962 Watson and
Crick received the
Nobel Prize for their
work.
• Note each of these pairs is one purine always
bonding to one pyrimidine.
• In a DNA molecule, A always pairs with T (=AT)
and G always pairs with C (= GC).
• You must know these pairs. Remember the pairs
as GoCAT
• The result of this pairing is the DNA double
helix.
• This massive molecule resembles a spiral
staircase.
• The 2 DNA strands of the double helix are
held together by hydrogen bonds
between the complementary base pairs.
• Each Adenine and Thymine base pair is
held together by 2 hydrogen bonds (A=T).
• Each Cytosine and Guanine base pair is
held together by 3 hydrogen bonds (C G)
• The number of base pairs for each
complete twist of helix is 10.
• Each one of the 2 strands has an end
called the 5’ (5 prime) and a 3’ (3 prime)
end.
• The numbers 5 and 3 come from the
number of the carbon atom in the pentose
sugar to which the phosphate group is
attached.
 5’
2’ 3’
4’ 1’ 1’ 4’
3’ 2’ 5’

5’ 2’ 3’
4’ 1’ 4’
1’
3’ 2’ 5’
• Double-stranded DNA twists into an –helix with two topological
features: a minor groove and a major groove.
• The two strands of DNA appear as ridges, separated by a trough.
These contours between two strands compose the minor groove.
• The major groove results from the twisting pattern of the -helix.
• Every 10 base pairs, a distance of about 3.6 nm, the helix completes
a full turn, forming the major groove that resembles a saddle.
• Variations in nucleotide sequence cause subtle regional alterations
in the shape of DNA and the topology of the major and minor
grooves.
• This structural variation is information that is used by the DNA-
binding proteins to attach to the correct location to regulate
expression of specific genes.
• The DNA in animal cells is highly compressed
into tight structures with the aid of DNA-binding
proteins called histones.
• The long strands of DNA wrap twice around the
barrel-shaped histones until a structure
resembling a strand of pearls is formed.
• These strands are then twisted and folded into
highly compressed arrangements.
• DNA twisting and folding has two main advantages to
cells.
• First, it allows the cell to fit large amounts of DNA into
the small volume.
• Second, coating DNA with histones helps reduce the
damage caused by radiation and chemicals.
• However, in this compressed configuration DNA is
biochemically inert; it cannot function as a template for
RNA synthesis (transcription) or DNA synthesis
(replication).
• Cells must use histone-modifying enzymes to release
histones from DNA, thereby regulating gene expression.
• The entire collection of DNA within a cell is called the
genome.
• Within the nucleus, the genome is divided into separate
segments of DNA called chromosomes.
• Within chromosomes are the genes, which possess the
DNA sequences that are used to produce all the different
types of RNA, including the mRNA that encodes
proteins.
• Each gene also possesses regions of DNA called
promoters that determine when the gene is expressed.
The Double Alpha Helix of DNA

When two strands of DNA

join to form the alpha
helix, it is due to
hydrogen bonding
between the
complimentary purine and
pyrimidine bases on each
complimentary strand.
Adenine forms hydrogen
bonds with Thymine and
Guanine forms hydrogen
bonds with Cytosine.
This is called
Complimentary Base
Pairing.
The complimentary
strands run in
opposite directions or
anti-parallel to each
other.
The strands begin to spiral and due to hydrogen
bonding takes on the double alpha helix form.
Other nucleotides are vital components
of energy reactions
• In addition to serving as subunits of DNA and RNA, nucleotide
bases play other critical roles in the life of a cell.
• For example, adenine is a key component of the molecule
adenosine triphosphate, the energy currency of the cell.
• Cells use ATP as energy in a variety of transactions, the way we use
money in society.
• ATP is used to drive energetically unfavorable chemical reactions, to
power transport across membranes, and to power the movement of
cells.
• Two other important nucleotide-containing molecules are
nicotinamide adenine dinucleotide (NAD+) and flavin adenine
• dinucleotide (FAD).
• These molecules function as electron carriers in a variety of cellular
processes.
Comparing and Contrasting
DNA and RNA
• DNA bases (A,T,G,C) • RNA bases (A,U,G,C)
• Deoxyribose sugar • Ribose sugar
• Original information for • Working copy for making
making proteins proteins
• One form or type • Variety of forms, m-RNA,
• Found primarily in the t-RNA, r-RNA
nucleus, forms • Found in nucleus and
chromosomes during cell throughout the cell
division • Smaller molecules (single
• Large molecule (double stranded)
stranded)
 ENZYMES
LESSON IV

BIO 1401

Dr Sydney Malama

Introduction

Enzymes are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical
reactions. The molecules upon which enzymes may act are called substrates, and the enzyme
converts the substrates into different molecules known as products. Almost all metabolic processes
in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic
pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called
enzymology and a new field of pseudo enzyme analysis has recently grown up, recognising that
during evolution, some enzymes have lost the ability to carry out biological catalysis, which is
often reflected in their amino acid sequences and unusual 'pseudo catalytic' properties. Enzymes
are known to catalyze more than 5,000 biochemical reaction types. Other biocatalysts are catalytic
RNA molecules, called ribozymes. Enzymes' specificity comes from their unique three-
dimensional structures.

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some
enzymes can make their conversion of substrate to product occur many millions of times faster.
An extreme example is orotidine 5'-phosphate decarboxylase, which allows a reaction that would
otherwise take millions of years to occur in milliseconds. Chemically, enzymes are like any
catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction.
Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be
affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators
are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors.
An enzyme's activity decreases markedly outside its optimal temperature and pH, and many
enzymes are (permanently) denatured when exposed to excessive heat, losing their structure and
catalytic properties.
Some enzymes are used commercially, for example, in the synthesis of antibiotics. Some
household products use enzymes to speed up chemical reactions: enzymes in biological washing
powders break down protein, starch or fat stains on clothes, and enzymes in meat tenderizer break
down proteins into smaller molecules, making the meat easier to chew.

Naming conventions

An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with
the word ending in -ase: Examples are lactase, alcohol dehydrogenase and DNA polymerase.
Different enzymes that catalyze the same chemical reaction are called isozymes.

The International Union of Biochemistry and Molecular Biology have developed a nomenclature
for enzymes, the EC numbers; each enzyme is described by a sequence of four numbers preceded
by "EC", which stands for "Enzyme Commission". The first number broadly classifies the enzyme
based on its mechanism.

The top-level classification is:

 EC 1, Oxidoreductases: catalyze oxidation/reduction reactions

 EC 2, Transferases: transfer a functional group (e.g. a methyl or phosphate group)
 EC 3, Hydrolases: catalyze the hydrolysis of various bonds
 EC 4, Lyases: cleave various bonds by means other than hydrolysis and oxidation
 EC 5, Isomerases: catalyze isomerization changes within a single molecule
 EC 6, Ligases: join two molecules with covalent bonds.

These sections are subdivided by other features such as the substrate, products, and chemical
mechanism. An enzyme is fully specified by four numerical designations. For example, hexokinase
(EC 2.7.1.1) is a transferase (EC 2) that adds a phosphate group (EC 2.7) to a hexose sugar, a
molecule containing an alcohol group (EC 2.7.1).

Structure
 Enzyme activity initially increases with temperature (Q10 coefficient) until the enzyme's
structure unfolds (denaturation), leading to an optimal rate of reaction at an intermediate
temperature.

Enzymes are generally globular proteins, acting alone or in larger complexes. The sequence of the
amino acids specifies the structure which in turn determines the catalytic activity of the enzyme.
Although structure determines function, a novel enzymatic activity cannot yet be predicted from
structure alone. Enzyme structures unfold (denature) when heated or exposed to chemical
denaturants and this disruption to the structure typically causes a loss of activity. Enzyme
denaturation is normally linked to temperatures above a species' normal level; as a result, enzymes
from bacteria living in volcanic environments such as hot springs are prized by industrial users for
their ability to function at high temperatures, allowing enzyme-catalysed reactions to be operated
at a very high rate.

Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid
residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal
fatty acid synthase. Only a small portion of their structure (around 2–4 amino acids) is directly
involved in catalysis: the catalytic site. This catalytic site is located next to one or more binding
sites where residues orient the substrates. The catalytic site and binding site together compose the
enzyme's active site. The remaining majority of the enzyme structure serves to maintain the precise
orientation and dynamics of the active site.

In some enzymes, no amino acids are directly involved in catalysis; instead, the enzyme contains
sites to bind and orient catalytic cofactors. Enzyme structures may also contain allosteric sites
where the binding of a small molecule causes a conformational change that increases or decreases
activity.
A small number of RNA-based biological catalysts called ribozymes exist, which again can act
alone or in complex with proteins. The most common of these is the ribosome which is a complex
of protein and catalytic RNA components.

Mechanism

Organisation of enzyme structure and lysozyme example. Binding sites in blue, catalytic site in
red and peptidoglycan substrate in black

Substrate binding

Enzymes must bind their substrates before they can catalyse any chemical reaction. Enzymes are
usually very specific as to what substrates they bind and then the chemical reaction catalysed.
Specificity is achieved by binding pockets with complementary shape, charge and
hydrophilic/hydrophobic characteristics to the substrates. Enzymes can therefore distinguish
between very similar substrate molecules to be chemoselective, regioselective and stereospecific.

Some of the enzymes showing the highest specificity and accuracy are involved in the copying
and expression of the genome. Some of these enzymes have "proof-reading" mechanisms. Here,
an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the
product is correct in a second step This two-step process results in average error rates of less than
1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading
mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes.
Conversely, some enzymes display enzyme promiscuity, having broad specificity and acting on a
range of different physiologically relevant substrates. Many enzymes possess small side activities
which arose fortuitously (i.e. neutrally), which may be the starting point for the evolutionary
selection of a new function.

Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex.
Hexokinase has a large induced fit motion that closes over the substrates adenosine triphosphate
and xylose. Binding sites in blue, substrates in black and Mg2+ cofactor in yellow.

"Lock and key" model

To explain the observed specificity of enzymes, in 1894 Emil Fischer proposed that both the
enzyme and the substrate possess specific complementary geometric shapes that fit exactly into
one another. This is often referred to as "the lock and key" model. This early model explains
enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve.
Induced fit model

In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are
rather flexible structures, the active site is continuously reshaped by interactions with the substrate
as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid
active site; the amino acid side-chains that make up the active site are molded into the precise
positions that enable the enzyme to perform its catalytic function. In some cases, such as
glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The
active site continues to change until the substrate is completely bound, at which point the final
shape and charge distribution is determined. Induced fit may enhance the fidelity of molecular
recognition in the presence of competition and noise via the conformational proofreading
mechanism.

Allosteric modulation

Allosteric sites are pockets on the enzyme, distinct from the active site, that bind to molecules in
the cellular environment. These molecules then cause a change in the conformation or dynamics
of the enzyme that is transduced to the active site and thus affects the reaction rate of the enzyme.
In this way, allosteric interactions can either inhibit or activate enzymes. Allosteric interactions
with metabolites upstream or downstream in an enzyme's metabolic pathway cause feedback
regulation, altering the activity of the enzyme according to the flux through the rest of the pathway.

Cofactors
Chemical structure for thiamine pyrophosphate and protein structure of transketolase. Thiamine
pyrophosphate cofactor in yellow and xylulose 5-phosphate substrate in black.

Some enzymes do not need additional components to show full activity. Others require non-protein
molecules called cofactors to be bound for activity.] Cofactors can be either inorganic (e.g., metal
ions and iron-sulfur clusters) or organic compounds (e.g., flavin and heme). These cofactors serve
many purposes; for instance, metal ions can help in stabilizing nucleophilic species within the
active site. Organic cofactors can be either coenzymes, which are released from the enzyme's
active site during the reaction, or prosthetic groups, which are tightly bound to an enzyme. Organic
prosthetic groups can be covalently bound (e.g., biotin in enzymes such as pyruvate carboxylase)

An example of an enzyme that contains a cofactor is carbonic anhydrase, which uses a zinc
cofactor bound as part of its active site. These tightly bound ions or molecules are usually found
in the active site and are involved in catalysis. For example, flavin and heme cofactors are often
involved in redox reactions.

Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins.
An enzyme together with the cofactor(s) required for activity is called a holoenzyme (or
haloenzyme). The term holoenzyme can also be applied to enzymes that contain multiple protein
subunits, such as the DNA polymerases; here the holoenzyme is the complete complex containing
all the subunits needed for activity.

Coenzymes

Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme.
Coenzymes transport chemical groups from one enzyme to another. Examples include NADH,
NADPH and adenosine triphosphate (ATP). Some coenzymes, such as flavin mononucleotide
(FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP), and tetrahydrofolate
(THF), are derived from vitamins. These coenzymes cannot be synthesized by the body de novo
and closely related compounds (vitamins) must be acquired from the diet. The chemical groups
carried include:

 the hydride ion (H−), carried by NAD or NADP+

 the phosphate group, carried by adenosine triphosphate
 the acetyl group, carried by coenzyme A
 formyl, methenyl or methyl groups, carried by folic acid and
 the methyl group, carried by S-adenosylmethionine
 Since coenzymes are chemically changed as a consequence of enzyme action, it is useful
to consider coenzymes to be a special class of substrates, or second substrates, which are
common to many different enzymes. For example, about 1000 enzymes are known to use
the coenzyme NADH.

Coenzymes are usually continuously regenerated and their concentrations maintained at a steady
level inside the cell. For example, NADPH is regenerated through the pentose phosphate pathway
and S-adenosylmethionine by methionine adenosyltransferase. This continuous regeneration
means that small amounts of coenzymes can be used very intensively. For example, the human
body turns over its own weight in ATP each day.
Thermodynamics

The energies of the stages of a chemical reaction. Uncatalysed (dashed line), substrates need a lot
of activation energy to reach a transition state, which then decays into lower-energy products.
When enzyme catalysed (solid line), the enzyme binds the substrates (ES), then stabilizes the
transition state (ES‡) to reduce the activation energy required to produce products (EP) which are
finally released.

As with all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction.
In the presence of an enzyme, the reaction runs in the same direction as it would without the
enzyme, just more quickly. For example, carbonic anhydrase catalyzes its reaction in either
direction depending on the concentration of its reactants:

(1)
(in tissues; high CO2 concentration)

(2)
(in lungs; low CO2 concentration)

The rate of a reaction is dependent on the activation energy needed to form the transition state
which then decays into products. Enzymes increase reaction rates by lowering the energy of the
transition state. First, binding forms a low energy enzyme-substrate complex (ES). Second, the
enzyme stabilises the transition state such that it requires less energy to achieve compared to the
uncatalyzed reaction (ES‡). Finally, the enzyme-product complex (EP) dissociates to release the
products.

Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be
used to "drive" a thermodynamically unfavourable one so that the combined energy of the products
is lower than the substrates. For example, the hydrolysis of ATP is often used to drive other
chemical reactions.

Kinetics

A chemical reaction mechanism with or without enzyme catalysis. The enzyme (E) binds
substrate (S) to produce product (P).

Saturation curve for an enzyme reaction showing the relation between the substrate concentration
and reaction rate.

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products.
The rate data used in kinetic analyses are commonly obtained from enzyme assays. In 1913 Leonor
Michaelis and Maud Leonora Menten proposed a quantitative theory of enzyme kinetics, which is
referred to as Michaelis–Menten kinetics. The major contribution of Michaelis and Menten was to
think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme,
forming the enzyme-substrate complex. This is sometimes called the Michaelis–Menten complex
in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the
product. This work was further developed by G. E. Briggs and J. B. S. Haldane, who derived
kinetic equations that are still widely used today.

Enzyme rates depend on solution conditions and substrate concentration. To find the maximum
speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of
product formation is seen. This is shown in the saturation curve on the right. Saturation happens
because, as substrate concentration increases, more and more of the free enzyme is converted into
the substrate-bound ES complex. At the maximum reaction rate (Vmax) of the enzyme, all the
enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total
amount of enzyme.

Vmax is only one of several important kinetic parameters. The amount of substrate needed to achieve
a given rate of reaction is also important. This is given by the Michaelis–Menten constant (Km),
which is the substrate concentration required for an enzyme to reach one-half its maximum reaction
rate; generally, each enzyme has a characteristic KM for a given substrate. Another useful constant
is kcat, also called the turnover number, which is the number of substrate molecules handled by one
active site per second.

The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity
constant and incorporates the rate constants for all steps in the reaction up to and including the first
irreversible step. Because the specificity constant reflects both affinity and catalytic ability, it is
useful for comparing different enzymes against each other, or the same enzyme with different
substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is
about 108 to 109 (M−1 s−1). At this point every collision of the enzyme with its substrate will result
in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion
rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of
such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase,
fumarase, β-lactamase, and superoxide dismutase. Michaelis–Menten kinetics relies on the law of
mass action, which is derived from the assumptions of free diffusion and thermodynamically
driven random collision. Many biochemical or cellular processes deviate significantly from these
conditions, because of macromolecular crowding and constrained molecular movement. More
recent, complex extensions of the model attempt to correct for these effects.
Inhibition

An enzyme binding site that would normally bind substrate can alternatively bind a competitive
inhibitor, preventing substrate access. Dihydrofolate reductase is inhibited by methotrexate which
prevents binding of its substrate, folic acid. Binding site in blue, inhibitor in green, and substrate
in black. (PDB: 4QI9)

The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in
structure (differences show in green). As a result, methotrexate is a competitive inhibitor of many
enzymes that use folates.

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

Types of inhibition
Competitive

A competitive inhibitor and substrate cannot bind to the enzyme at the same time. Often
competitive inhibitors strongly resemble the real substrate of the enzyme. For example, the drug
methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the
reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of
dihydrofolate and this drug are shown in the accompanying figure. This type of inhibition can be
overcome with high substrate concentration. In some cases, the inhibitor can bind to a site other
than the binding-site of the usual substrate and exert an allosteric effect to change the shape of the
usual binding-site.

Non-competitive

A non-competitive inhibitor binds to a site other than where the substrate binds. The substrate still
binds with its usual affinity and hence Km remains the same. However, the inhibitor reduces the
catalytic efficiency of the enzyme so that Vmax is reduced. In contrast to competitive inhibition,
non-competitive inhibition cannot be overcome with high substrate concentration.

Uncompetitive

An uncompetitive inhibitor cannot bind to the free enzyme, only to the enzyme-substrate complex;
hence, these types of inhibitors are most effective at high substrate concentration. In the presence
of the inhibitor, the enzyme-substrate complex is inactive. This type of inhibition is rare.

Mixed

A mixed inhibitor binds to an allosteric site and the binding of the substrate and the inhibitor affect
each other. The enzyme's function is reduced but not eliminated when bound to the inhibitor. This
type of inhibitor does not follow the Michaelis–Menten equation.

Irreversible

An irreversible inhibitor permanently inactivates the enzyme, usually by forming a covalent bond
to the protein. Penicillin and aspirin are common drugs that act in this manner.
Functions of inhibitors

In many organisms, inhibitors may act as part of a feedback mechanism. If an enzyme produces
too much of one substance in the organism, that substance may act as an inhibitor for the enzyme
at the beginning of the pathway that produces it, causing production of the substance to slow down
or stop when there is sufficient amount. This is a form of negative feedback. Major metabolic
pathways such as the citric acid cycle make use of this mechanism. Since inhibitors modulate the
function of enzymes they are often used as drugs. Many such drugs are reversible competitive
inhibitors that resemble the enzyme's native substrate, similar to methotrexate above; other well-
known examples include statins used to treat high cholesterol, and protease inhibitors used to treat
retroviral infections such as HIV. A common example of an irreversible inhibitor that is used as a
drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation
messenger prostaglandin. Other enzyme inhibitors are poisons. For example, the poison cyanide
is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the
enzyme cytochrome c oxidase and blocks cellular respiration.

Factors affecting enzyme activity

As enzymes are made up of proteins, their actions are sensitive to change in many physio chemical
factors such as pH, temperature, substrate concentration, etc.

The following table shows pH optima for various enzymes.

Enzyme Optimum pH pH description

Pepsin 1.5–1.6 Highly acidic
Invertase 4.5 Acidic
Lipase (stomach) 4.0–5.0 Acidic
Lipase (castor oil) 4.7 Acidic
Lipase (pancreas) 8.0 Alkaline
Amylase (malt) 4.6–5.2 Acidic
Amylase (pancreas) 6.7–7.0 Acidic-neutral
Enzyme Optimum pH pH description
Cellobiase 5.0 Acidic
Maltase 6.1–6.8 Acidic
Sucrase 6.2 Acidic
Catalase 7.0 Neutral
Urease 7.0 Neutral
Cholinesterase 7.0 Neutral
Ribonuclease 7.0–7.5 Neutral
Fumarase 7.8 Alkaline
Trypsin 7.8–8.7 Alkaline
Adenosine triphosphate 9.0 Alkaline
Arginase 10.0 Highly alkaline

Biological function

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for
signal transduction and cell regulation, often via kinases and phosphatases. They also generate
movement, with myosin hydrolyzing ATP to generate muscle contraction, and also transport cargo
around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps
involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase
generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV
integrase and reverse transcriptase, or for viral release from cells, like the influenza virus
neuraminidase.

An important function of enzymes is in the digestive systems of animals. Enzymes such as

amylases and proteases break down large molecules (starch or proteins, respectively) into smaller
ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be
absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such
as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different
food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce
another enzyme, cellulase, to break down the cellulose cell walls of plant fiber.

Metabolism

The metabolic pathway of glycolysis releases energy by converting glucose to pyruvate via a
series of intermediate metabolites. Each chemical modification (red box) is performed by a
different enzyme.

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic
pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic
reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can
catalyze the same reaction in parallel; this can allow more complex regulation: with, for example,
a low constant activity provided by one enzyme but an inducible high activity from a second
enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would
neither progress through the same steps and could not be regulated to serve the needs of the cell.
Most central metabolic pathways are regulated at a few key steps, typically through enzymes
whose activity involves the hydrolysis of ATP. Because this reaction releases so much energy,
other reactions that are thermodynamically unfavorable can be coupled to ATP hydrolysis, driving
the overall series of linked metabolic reactions.

Control of activity
There are five main ways that enzyme activity is controlled in the cell.

Regulation

Enzymes can be either activated or inhibited by other molecules. For example, the end product(s)
of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually
the first irreversible step, called committed step), thus regulating the amount of end product made
by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because
the amount of the end product produced is regulated by its own concentration. Negative feedback
mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the
demands of the cells. This helps with effective allocations of materials and energy economy, and
it prevents the excess manufacture of end products. Like other homeostatic devices, the control of
enzymatic action helps to maintain a stable internal environment in living organisms.

Post-translational modification

Examples of post-translational modification include phosphorylation, myristoylation and

glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes,
including glycogen synthase, helps control the synthesis or degradation of glycogen and allows
the cell to respond to changes in blood sugar. Another example of post-translational modification
is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in
inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach
where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it
enters the gut. This type of inactive precursor to an enzyme is known as a zymogenor proenzyme.

Quantity

Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished
by a cell in response to changes in the cell's environment. This form of gene regulation is called
enzyme induction. For example, bacteria may become resistant to antibiotics such as penicillin
because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring
within the penicillin molecule. Another example comes from enzymes in the liver called
cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of
these enzymes can cause drug interactions. Enzyme levels can also be regulated by changing the
rate of enzyme degradation. The opposite of enzyme induction is enzyme repression.

Subcellular distribution

Enzymes can be compartmentalized, with different metabolic pathways occurring in different

cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the
cytosol, endoplasmic reticulum and Golgi and used by a different set of enzymes as a source of
energy in the mitochondrion, through β-oxidation. In addition, trafficking of the enzyme to
different compartments may change the degree of protonation (e.g., the neutral cytoplasm and the
acidic lysosome) or oxidative state (e.g., oxidizing periplasm or reducing cytoplasm) which in turn
affects enzyme activity In contrast to partitioning into membrane bound organelles, enzyme
subcellular localisation may also be altered through polymerisation of enzymes into
macromolecular cytoplasmic filaments.
TOPIC 2: DNA REPLICATION

2.1 Introduction
DNA is a biomolecule which is capable of reproducing itself in a process called replication.
Watson and Crick proposed that during replication the two DNA strands were capable of
separating and acting as templates to which a complementary set of nucleotides would attach
by base pairing to form new DNA strands.

2.2 Objectives: At the end of this unit you should be able to:
(i) Prove that DNA is capable of accurately reproducing itself.
(ii) Explain the semi-conservative mechanism of DNA replication with reference to the
experiments of Meselson and Stahl.
(iii) List the enzymes and proteins that are involved in prokaryotic DNA replication and
explain
the role of each of them.
(iv) Explain the different stages of prokaryotic DNA replication
(v) Differentiate between the synthesis of the leading strand and that of the lagging strand

2.3 Semi-conservative mechanism of DNA replication: The replication of DNA is semi-

conservative in which the original double helix molecule is duplicated into two identical
copies. Each DNA copy contains one old strand and one new strand. Evidence of this
mechanism of DNA replication came from the experiment by Meselson and Stahl who used
E.coli cells.

The cells were initially grown in a nutrient medium containing ‘heavy’ nitrogen isotope (15N)
15
for many generations until all the nitrogen in their DNA contained N. The cells were then
transferred to a medium containing the ‘light’ nitrogen isotope (14N) and different generations
of bacterial cells were sampled from the medium. The DNA in each sample was analysed by
density-gradient equilibrium centrifugation using caesium chloride (CsCl). In this method
different concentrations of CsCl were added to a centrifuge tube. The most concentrated
solution was added first followed by less concentrated solutions and the most dilute solution
was added last. This CsCl solution gradient is able to separate heavy-heavy (HH), light-light
(LL) and heavy-light (HL) DNA double helices into separate bands. The bands which were
observed in the experiment proved that DNA undergoes the semi-conservative replication
and not the conservative replication. The semi-conservative type of DNA replication has also
been conformed in eukaryotic cells
Figure 2.1.The Meselson-Stahl experiment. (a) Cells were grown for many generations in a medium containing
only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N as shown by a single band (blue) when
centrifuged in a CsCl density gradient. (b) Once the cells had been transferred to a medium containing only light
nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient
(purple band). (c) Continuation of replication for a second generation yielded two hybrid DNAs and two light
DNAs (red), confirming semi conservative replication. Adapted from Griffiths et al. 1993. An introduction to
Genetic Analysis. Page 316.

2.4 DNA replication process in prokaryotes

DNA replication in prokaryote is well represented by E.coli. The process involves the
following major steps: (i) Replication initiation, (ii) DNA denaturation – melting –unzipping,
(iii) Priming, (iv) Elongation - DNA synthesis at growing fork (v) Separation of circular
daughter molecules, (vi) Proof reading.
2.4.1 Initiation of replication in E.coli

The replication of DNA requires the removal of the supercoils and the attachment of enzymes
and other proteins to the origin of replication on the DNA molecule. The supercoils are
removed by the enzyme called topoisomerase followed by the attachment of a protein called
DnaA. This results in the formation of the initiation complex.

Figure 2.2. Initiation of DNA replication at E.coli origin of replication.

2.4.2 Double stranded DNA denaturation

This process is also called DNA melting or unzipping. When DnaA binds to the double helix,
the first separation of the two strands takes place. This process requires ATP. This is
followed by the binding of the enzyme DNA helicase which leads to the separation of more
base pairs. The helicase moves along the DNA double helix using the energy from ATP
hydrolysis to separate the two strands. The denaturation of double stranded DNA is in the 5′
→ 3′ direction. The single strand binding protein prevents the double helix from rejoining.
Figure 2.3. DNA denaturation by the action of helicase in E.coli.

2.4.3 Formation of RNA primers in E.coli

This process is called priming. It is well known that DNA polymerase cannot initiate a new
DNA chain but can only elongate a pre-existing DNA or RNA strand. Therefore, before the
parental DNA is replicated, a short RNA molecule, complementary the DNA strand, is
synthesised by RNA polymerase. This RNA molecule is called a primer and it attaches itself
to the initiation complex. A primer is synthesised on each DNA strand in the 5′→3′ direction.

Figure 2.4. Priming on the leading and lagging strand DNA templates by the action of
primase in E.coli.
2.4.4 DNA synthesis - addition of nucleotides at the growing fork by E. coli DNA
This is a polymerization reaction catalysed by DNA polymerase. Although the two strands of
DNA double helix are anti-parallel, DNA polymerase catalyses nucleotide addition at the 3’-
hydroxyl end of each growing chain. Therefore each strand is elongated only in the 5′→3′
direction. Synthesis of a new DNA strand involves the addition of deoxyribonucleotide
triphosphates (dNTPs) which include: dATP, dGTP, dTTP, and dCTP.

2.4.4.1 Leading strand synthesis: At each growing fork, one DNA strand, called the leading
strand, is synthesized continuously from a single primer on the leading strand template. The
leading strand grows in the 5’→3’ direction, like the growing fork.

2.4.4.2 Lagging strand synthesis: Synthesis of the lagging strand is more complicated,
because DNA polymerases can add nucleotides only to the 3’ end of a primer or growing
DNA strand. Movement of the growing fork exposes the lagging-strand template on which
short RNA primers are copied. Each of these primers is then elongated by addition of dNTPs
to its 3′ end. . In E.coli, this reaction is catalysed by DNA polymerase III (poly III). The
resulting short fragments containing RNA covalently linked to DNA are called Okazaki
fragments. In bacteria and bacteriophage, Okazaki fragments contain 1000-2000 nucleotides.
The overall direction of growth of the lagging strand is from its 3′→5′ end, complementary to
the polarity of its template but opposite to the direction of nucleotide addition by DNA
polymerases. DNA polymerase I is primarily involved in removing RNA primers from
Okazaki fragments (exonuclease activity) and filling the resultant gaps using its 5′→3′
polymerizing activity. DNA ligase joins adjacent completed Okazaki fragments.

DNA polymerase II functions in the inducible SOS response and also fills gaps and appears
to facilitate DNA synthesis directed by damaged templates. DNA polymerase II resembles
DNA polymerase I in its activity, but is a DNA repair enzyme, bringing about the growth in
5′→3′ direction using free 3′- OH groups.
Figure 2.5. DNA replication at a growing fork. Source: Lodish et al. 2000. Molecular Cell
Biology, 4th edition. Page 113.

2.4.5 Proof reading by DNA polymerase

Occasionally, a wrong base is inserted during DNA synthesis. This is corrected by the
proofreading function of all bacterial polymerases.

2.4.6 Separation of circular daughter molecules

During DNA replication the parental strands remain intact and retain their superhelicity. This
poses stearic and topological constraints to the completion of the replication of a circular
DNA molecule as the two growing forks approach each other. In E.coli, decatenation of the
two daughter cells is catalyzed by Topo II (DNA gyrase) and topoisomerase IV (Topo IV).
This helps separate the daughter DNA molecules from each other
Table 2.1 Enzymes involved in prokaryotic DNA replication
# Enzyme Function
1 DnaA protein Binds DNA to form the initiation complex
2 Topoisomerase Removes coils and supercoils from DNA
3 DNA polymerase I Removes primers from the new DNA strand
4 DNA polymerase II Repairs mistakes during DNA polymerization and also fills in
the gaps left after the removal of primers from lagging strand
5 DNA polymerase III DNA polymerization
6 Helicase DNA denaturation
7 Primase Synthesis of short RNA primers on template DNA
8 Ligase Joins DNA nucleotides through covalent bonds
9 Single strand binding protein Prevents single stranded DNA from forming double strands

2.5 Revision questions: DNA replication

1. Outline the central dogma of molecular biology.

2. Explain the experiments which provided evidence in support of the mechanism of DNA
replication.
3. Differentiate between conservative and semi-conservative mechanisms of DNA
replication.
4. Describe the various stages of prokaryotic DNA replication.
5. List the enzymes and non-enzyme proteins involved in prokaryotic DNA synthesis and
explain
the role of each of them.
6. Differentiate between leading and lagging strand synthesis of prokaryotic DNA.
7. Explain how accuracy is achieved during DNA replication.

2.6 Summary: DNA replication

The DNA molecule is able to replicate itself and this is the basis of growth and reproduction among
living organisms. DNA replicates semi-conservatively with the leading strand template
replicating continuously and the lagging strand template replicating discontinuously. The
steps involved in DNA replication are (i) Replication initiation, (ii) DNA denaturation –
melting –unzipping, (iii) Priming, (iv) Elongation - DNA synthesis at growing fork (v)
Separation of circular daughter molecules, (vi) Proof reading. All these steps are controlled
by specific enzymes.
TOPIC 2: TRANSCRIPTION

2.1 Introduction

Transcription is the synthesis of RNA using information from DNA. This process ensures that
synthesis of proteins using information from DNA is properly regulated. Because of this
intermediary process, proteins are synthesised in the right place in the right amounts at the right
time.

2.2 Objectives: At the end of this topic you should be able to:
1. To explain the process of transcription.
2. To describe the phases of initiation, elongation and termination.
3. To discuss posttranscriptional modification of eukaryotic mRNA, tRNA and rRNA.

2.3 Mechanism of transcription

Transcription involves the following stages: (i) initiation (ii) elongation (iii) termination and (iv)
the processing of primary transcript products into mature RNA molecules.
2.3.1 Initiation
Before transcription can take place, the DNA double helix of the gene to be transcribed has to
unwind and the two DNA polynucleotide chains have to separate (unzip) in order to expose the
nucleotide bases. This is done by the helicase activity of RNA polymerase following the binding
of RNA polymerase to DNA at a region called promoter. During the initiation phase of
transcription, the first two nucleotides of the RNA (usually A and C) are joined together
(transcribed).

Figure 2.1. Initiation of RNA transcription

2.3.2 Elongation

The elongation phase begins when RNA polymerase moves along the template DNA strand
binding one deoxynucleotide at a time as it moves. RNA polymerase adds ribonucleotides
opposite the DNA templated on the 3′- end of the growing RNA chain. The new RNA therefore
grows in the 5′ → 3′ direction. The front part of the RNA polymerase separates the two DNA
strands as it moves along.

Figure 2.2. Elongation during RNA synthesis/transcription

2.3.3 Termination of transcription

There are two types of transcription termination mechanisms.
(i) The first mechanism which is referred to as rho (ρ)-independent termination of
transcription, occurs at specific base sequences called palindromic sequences in the
RNA molecule (AAAGGCUCC-UUUU-GGAGCCUUU). When the palindromic
sequence is transcribed, it forms a hair-pin loop which disturbs the RNA polymerase and
results in termination of transcription;

Figure 2.3 Rho-independent termination of transcription

 (ii) the second mechanism is the (rho) ρ-dependent termination which uses the ρ factor to
stop RNA synthesis at specific sites. When the RNA polymerase reaches certain sites of
DNA the rho factor approaches the new mRNA and hooks it up thereby disturbing the
RNA polymerase and causing the termination of the transcription process.

2.3.4 Posttranscriptional modification of newly synthesized RNAs

The newly synthesized RNAs (mRNA, tRNA and rRNA) are called precursor molecules and
must be modified before they become mature and functional. Modification involves removal of
some nucleotides and transformation of other nucleotides.
2.3.4.1 Messenger RNA posttranscriptional modification
Precursor mRNA undergoes capping, polyadenylation and splicing before it becomes mature
mRNA.
Capping is the addition of methylated guanosine head on the 5´ end: The cap protects mRNA
from degradation and helps in nuclear export and ribosome binding.
Polyadenylation is the addition of poly (A) tail on the 3´ end: The poly-A tail protects mRNA
from degradation and aid in the export of mRNA from the nucleus; also aids in transcription
termination and in translation.
Splicing is the removal of introns and joining of exons together.

Importance of mRNA posttranscriptional modification

• Splicing: Helps in regulating gene expression because transcripts cannot leave the nucleus
to be translated until their introns are removed.
• The 5′ cap: Protects the nascent mRNA from degradation and assists in ribosome binding
during translation.
• The poly(A) tail: Protects the mRNA molecule from enzymatic degradation in the
cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and
translation
Figure 2.4 Messenger RNA post-transcription modification

2.3.4.2. Ribosomal RNA posttranscriptional modification

Ribosomal RNA (rRNA) is synthesised as precursor rRNA. Precursor rRNA undergoes
methylation and shortening to form mature rRNA. Mature rRNA combines with protein to
produce ribosome. A complete ribosome has two subunits. The prokaryotic ribosome is 70s (30s
+ 50s) while the eukaryotic ribosome is 80s (60s +40s).

Figure 2.5 Formation of prokaryotic (a) and eukaryotic (b) ribosomes.

2.3.4.3. Transfer RNA posttranscriptional modification
Precursor tRNA is converted into cloverleaf shaped mature tRNA which has the following
features: (i) D arm, (ii) T arm, (iii) anticodon and (iv) amino acid acceptor arm.

Figure 2.6 Mature tRNA

2.4 Transcription inhibiting drugs

Transcription inhibitors can be used as antibiotics against, for example, pathogenic bacteria
(antibacterial drugs) and fungi (antifungals). Rifampicin is an antibacterial drug which inhibits
prokaryotic DNA transcription while 8-Hydroxyquinoline is an antifungal drug which is a
transcription inhibitor.

2.5 REVIEW QUESTIONS

1. Explain differences between DNA replication and transcription.

2. Explain why the DNA molecule is referred to as the blue print for protein synthesis.
3. Explain the role of the promoter site during transcription.
4. Discuss posttranscriptional modification of RNA.
5. Differentiate between introns and exons and mention the type of RNA in which they
are found.
6. Describe the structure of each mature and functional prokaryotic RNA.
7. Explain the role of each RNA.

2.6 Summary
The synthesis of RNA using information from DNA is called transcription. The RNA molecule
is intermediate between DNA and proteins. The transcription process involves initiation,
elongation and termination. The stretch of DNA that is transcribed into an RNA molecule is
called a transcription unit. The section of DNA that holds the information for one polypeptide is
called a gene. Only one of the DNA strands is used as a template for the synthesis of RNA. This
is called the sense DNA strand. The other strand which is not transcribed is called the anti-sense
and it has the same code as the RNA transcript except for T in place of U.
TOPIC 3: TRANSLATION
3.1 Introduction
Translation, also called protein synthesis, is a process in which amino acids are assembled into
polypeptides based on the genetic information carried by mRNA derived from DNA. Translation
involves the participation of mRNA, tRNA and rRNA and a number of enzymes and other
proteins. The length of the polypeptide synthesised depends on the genetic information
(message) carried by the mRNA.

3.2 Objectives: At the end of this topic you should be able to:

1. To explain the role of mRNA and the genetic code

2. To explain the roles of tRNA and rRNA
3. To describe the mechanism of translation
4. To discuss posttranslational modification of newly synthesized polypeptides

3.3. Role of Messenger RNA

The mRNA carries the genetic code (triplet codon) derived from the template DNA. Messenger
RNA is a linear molecule which is a copy of the template DNA strand and varies in length
according to the length of the polypeptide that it codes for.
3.3.1 The genetic code
The genetic code is the whole sequence of nucleotides on the mRNA which is derived from the
DNA. The characteristics of the genetic code are; (i) It has codons in which the nucleotides of
mRNA are arranged as a linear sequence of successive triplets, (ii) it is non-overlapping meaning
that during translation, the codons do not overlap but are read sequentially, three at a time, (iii) it
is comaless meaning that all the nucleotides are used to code for amino acids with none of them
used for punctuation, (v) it is universal meaning that it is the same for all organisms ranging from
virus to humans, (v) it has polarity meaning that it is always read in the 5’ → 3’ direction and
(vi) it is degenerate meaning that more than one codon may specify the same amino acid. Except
tryptophan and methionine which have a single codon each, all the other 18 amino acids have
more than one codon (vii) There are three codons that are not recognized by any tRNA: UAA,
UAG, and UGA. These are termed nonsense codons or stop codons, because they signal protein
synthesis to stop at that point.

3.3.2 Wobble hypothesis

The interaction between the codon in the mRNA and the anticodon in the tRNA needs to be
exact in first two nucleotide positions, but does not have to be so in the third position. Non-
standard base-pairing might occur between the third bases of the anticodon and of the codon. The
degenerate base (third base) is said to be in the wobble position.
1
Table 3.1 Table of the genetic code. Source: Jones and Karp. 1994. Introducing genetics. Page
200.

3.3.3 Role of Ribosomal RNA

Ribosomal RNA (rRNA) makes up nearly 80% of the total RNA of the cell. It combines with
protein to form organelles called ribosomes. Ribosomes are the site of proteinsynthesis. Each
ribosome has two subunits; a small and a large subunit. The function of the ribosome is to hold
in position the mRNA, tRNA and the associated enzymes involved in protein synthesis until a
peptide bond is formed between the adjacent amino acids. The ribosome has the A site
(aminoacyl site) and the P site (peptidyl site).

3.3.4 Role of transfer tRNA

Transfer RNA (tRNA) acts as an intermediate molecule between the triplet code of mRNA and
the amino acid sequence of the polypeptide chain. It is a small molecule with about 80
nucleotides. Each amino acid has its own tRNA which carries a triplet anticodon for the
particular amino acid. The anticodon is complementary to a specific codon on mRNA. The 3´
end of the tRNA carries the sequence 5´ -CCA-3´ which is used to attach a specific amino acid.

2
3.3.5 The mechanism of translation in prokaryotes
The main steps involved in translation may be summarised under the following headings: (i)
Binding of mRNA to ribosome (ii) amino acid activation (iii) attachment of amino acids to
specific tRNA (iv) polypeptide chain initiation (v) chain elongation (vi) chain termination (vii)
posttranslational modification.

3.3.5.1 Binding of mRNA to ribosome

The small (30S) ribosomal subunit binds to mRNA to form the 30S initiation complex. The
Shine Dalgarno (SD) sequence guides the mRNA to the binding site of the ribosome. This is
followed by the binding of the large (50S) subunit to the 30S initiation complex to make the 70S
initiation complex. Ribosomes are usually found in clusters linked together by one strand of
mRNA. This complex known as a polyribosome or polysome enables several copies of the same
polypeptide to be produced simultaneously.

Figure 31a. Binding of mRNA to prokaryotic ribosomal small (30S) subunit

Figure 3.1b. Binding of 50S to 30S initiation complex (A=Peptidyl site; A=aminoacyl site)

Figure 3.2. A polyribosome.

3
3.3.5.2Amino acid activation
Each target amino acid is activated by ATP using the enzyme aminoacyl-tRNA synthetase. There
is one enzyme for each amino acid.

Figure 3.3 Amino acid activation. Source: http://www.mdpi.com/ijms/ijms-15-

00401/article_deploy/html/images/ijms-15-00401f6-1024.png.

3.3.5.3 Attachment of activated amino acid to tRNA

The activated amino acid is then attached to its specific tRNA at the CCA end of the tRNA.
After this attachment, the tRNA complex is ready to transport the amino acid to the ribosome.

Figure 3.4. Attachment of amino acid to tRNA. Source: http://www.mdpi.com/ijms/ijms-15-

00401/article_deploy/html/images/ijms-15-00401f6-1024.png.

4
Figure 3.5a. Attachment of f-met to its tRNA

Figure 3.5b. Attachment of met to its tRNA

3.3.5.4 Polypeptide chain initiation

The tRNA with the anti-codon UAC for the codon AUG binds both amino acids - methionine
(met) and formyl methionine (f-met). Out of the two amino acids, f-met is the one carried to the
P site of the ribosome/mRNA complex to initiate the polypeptide chain. On the other hand, the
amino acid met is carried to the A site whenever the codon AUG reached the A site during the
process of protein synthesis. Special proteins called initiation factors are involved in chain
initiation.

Figure 3.6 Attachment of f-met-tRNA complex to the P site of the initiation complex.

3.3.5.4 Chain elongation

The second amino acid is brought to the A site by its tRNA. The ribosome then translocates to
the right by one codon, releasing the unloaded f-met. Meanwhile the second amino acid forms a
peptide bond with f-met by the action of the enzyme peptidyl transferase which is located in the

5
large subunit. The resulting dipeptide is carried by the second tRNA which is now located in the
P site.
Then the third amino acid is brought to the A site by its specific tRNA and it forms a peptide
bond with the dipeptide as the ribosome translocates to the right by one codon. The process is
repeated as more amino acids are added, one at a time, to the growing polypeptide chain as the
ribosome moves down the mRNA strand in the 5' → 3' direction of mRNA. Proteins called
elongation factors are involved in chain elongation.

Figure 3.7 Binding of the second amino acid to the A site of a ribosome. Source: Adapted
from Stansfield, 1991. Outline of Theory and problems of Genetics. Page 279.

6
Figure 3.8 Binding of the more amino acids to the growing peptide chain. Source: Adapted
from Stansfield, 1991. Outline of Theory and problems of Genetics. Page 279.
3.3.5.5 Chain termination
When one of the stop codons (UAA or UAG or UGA) reaches the A site, the synthesis of the
polypeptide chain stops. None of the tRNA molecules is able to bind to the stop codon but
instead special proteins called release factors bind to the stop codon. At this point the newly
made polypeptide chain is released and the ribosome is detached from the mRNA and split into
its subunits. The mRNA is broken down while the ribosomal subunits are recycled.
3.3.5.6 Post-translational modification
In order to achieve its biologically active form, the new polypeptide must fold into its proper
three-dimensional conformation. Before or after folding, the new polypeptide may undergo
enzymatic processing, including (i) removal of some amino acids; (ii) addition of functional
groups to certain amino acid; (iii) and attachment of oligosaccharides.
Insulin is an example of a polypeptide which undergoes post-translational modification. It is
synthesised as inactive pro-insulin and must be modified for it to become active insulin.

Figure 3.9 Formation of insulin from pro-insulin. Source: http://www.chemistrylearning.

com/humulin-synthetic-insulin.

3.4 REVISION QUESTIONS

1. Differentiate between the genetic code and a codon.

2. Define a gene.

7
3. Describe gene expression.

4. Explain the experiments carried out by Francis and his co-workers to confirm that the
genetic code is formed by triplet nucleotides.

5. Calculate the number of possible combinations that can arise from the triplet codon
system using the four nucleotides of RNA and comment on the significance of this
number.

6. Explain four features of the genetic code.

7. How many amino acids does each three-base triplet on the mRNA molecule code for?

8. State the start codon and mention the amino acid that it codes for.

9. What do the tree codons, UAA, UAG and UGA code for? Explain the role played by
these codons during protein synthesis.

10. State three differences between DNA replication and transcription.

11. Why is the DNA molecule referred to as the blue print for protein synthesis?

12. Discuss posttranscriptional modification.

13. What is the role of the promoter site during transcription?

14. Describe the structure of each mature prokaryotic RNA.

15. Explain the role of each prokaryotic RNA.

16. Differentiate between introns and exons and mention the type of RNA in which they
are found.

17. Which part of a ribosome contains a tRNA molecule that is attached to a growing
polypeptide chain?

18. During translation, an mRNA molecule has a codon with the triplet sequence 5´-
AUC-3´. (i) Write the anticodon sequence in a tRNA molecule that would pair with
this codon. (ii) Name the amino acid carried by this tRNA (iii) write the
corresponding sequence on the DNA from which the mRNA was transcribed..

19. What kind of bonds join amino acids in a polypeptide chain?

8
20. Discuss post translational modifications.

21. Differentiate the functions of the three types of RNA.

4.5 SUMMARY
Translation, also called protein synthesis, is a process in which amino acids are assembled into
polypeptides based on the genetic information carried by mRNA derived from DNA. Translation
involves the participation of mRNA, tRNA and rRNA and a number of enzymes and other
proteins. The length of the polypeptide synthesised depends on the genetic information
(message) carried by the mRNA. The main steps involved in translation may be summarised
under the following headings: (i) Binding of tRNA to ribosome (ii) amino acid activation (iii)
polypeptide chain initiation (iv) chain elongation (v) chain termination (vi) posttranslational
modification.

9
TOPIC 4: REGULATION OF GENE EXPRESSION

4.1 Introduction
Cells of organisms contain all the information required to make any of its proteins at any region
of the body and at any time of its life. However, the synthesis of proteins in these organisms has
to be controlled so that the right protein is made in the right amounts at the appropriate time.
Control of gene expression enables cells to produce certain enzymes only when their substrates
are present in the environment or to turn off the synthesis of other enzymes when their products
reach excessive concentrations. Regulation of gene expression conserves energy and important
raw materials such as amino acids. Control of gene expression can be done at any point of
protein synthesis. In prokaryotic cells, the most efficient control mechanism occurs at the
transcription stage. In eukaryotic cells, gene regulation is quite complex. Different transcription
factors can either up- or down-regulate transcription of target genes. The RNA resulting from
transcription represents the unprocessed transcript. After being processed, mRNA is translated
and translation is also subject to differential regulation. The newly formed polypeptide is also
subject to a variety of post-transcriptional modification e.g. targeting and degradation. Both
eukaryotes and prokaryotes can have expression of their genes regulated by feedback inhibition.
Up-regulation is a process in which an internal or external signal results in increased expression
of one or more genes resulting in increased protein synthesis. Down-regulation is a process
resulting in decreased gene and corresponding protein expression.

4.2 Objectives: At the end of this topic you should be able to:
(i) Explain the basis of regulation of gene expression.
(ii) Explain end-product inhibition.
(iii) Describe the structure and explain the function the Lac Operon system.

4.3 Inducible and repressible systems

An inducible system is normally ‘switched off’ except in the presence of an inducer molecule
that switches the system on and allows gene expression to take place. A repressible system is
normally ‘switched on’ except in the presence of a co-repressor molecule that suppresses gene
expression.

4.4 The Lac operon

The lac operon consists of three structural genes which is controlled by a regulator gene. It also
works in conjunction with a promoter sequence, an operator sequence and a terminator sequence.
The three structural genes of the lac operon are: (i) lacZ gene which encodes the enzyme β-
galactosidase (that breaks the disaccharide lactose into the monosaccharides glucose and
galactose), (ii) lacY gene which encodes the membrane-bound protein β-galactoside permease
(that pumps lactose into the cell) and (iii) lacA gene which encodes the enzyme β-galactoside
transacetylase (that modifies lactose).

The lac operon is required for the efficient transport and metabolism of lactose in Escherichia
coli and some related bacteria. In the absence of glucose, the cell can use lactose as an energy
source, but it must first produce the enzymes that are needed to mobilise and digest the lactose
(also called β-galactoside).

Figure 4.1 Breakdown of lactose into galactose and glucose.

The lac operon ensures that the cell uses energy in producing the three enzymes only when
necessary. It achieves this by using the repressor molecule, which stops the production of the
three enzymes in the absence of lactose. The repressor molecule has two active sites to which
either an inducer molecule (lactose) may be attached to ‘switch the operator gene on’ or a co-
repressor (glucose) molecule may be attached to ‘switch the operator gene off’. When the
operator gene is switched on, the structural genes carry out transcription of mRNA which is then
translated into polypeptides. When the operator gene is switched off, no mRNA is transcribed
and no polypeptides are synthesized.

Figure 4.2 The lac Operon in the repressed state. The repressor protein binds to the
operator site and prevents transcription. The genes z, y and a are therefore switched off.
The symbols are: i= regulator gene, p = promoter site, o =operator site, z, y and a =
structural genes, t = terminator sequence. Source: Jones and Karp, 1994. Introducing genetics
Page 239.
Figure 4.3. The lac Operon in the induced state. When lactose (the inducer) is present, it
alters the shape of the repressor which can then no longer bind to the operator. The genes
z, y and a are therefore transcribed. . The symbols are: i= regulator gene, p = promoter
site, o =operator site, z, y and a = structural genes, t = terminator sequence. Source: Jones
and Karp 1994. Introducing genetics. Page 239.

4.4.1 The lac Operon in the repressed state: The repressor protein binds to the operator site
and prevents transcription. The genes z, y and a are therefore switched off and hence are not
transcribed (Fig. 4.2).

4.4.2 The lac Operon in the induced state: When lactose (the inducer) is present, it alters the
shape of the repressor which can then no longer bind to the operator. The genes z, y and a are
therefore transcribed (Fig. 4.3).

4.4.3 End-production inhibition

When E. coli is grown on a glucose medium, the regulator gene produces a repressor molecule
which binds the glucose (co-repressor). This makes the repressor molecule active and causes it to
bind with the operator gene and switches the whole lac operon off. This is a case of end-product
inhibition or repression. It also referred to as a negative feedback control mechanism in which
the enzymes for glucose production (from lactose breakdown) are not synthesized because the
glucose (end-product) is already there.
4.5 Revision questions

1. Are all prokaryotic genes expressed all the time? Discuss.

2. How do prokaryotic cells switch-off mRNA synthesis when enzymes are not needed?

3. Eukaryotes do not employ a single, simple method of gene control. Explain.

4. What are inducible enzymes? When does a cell produce these enzymes?

5. Are cancer-causing genes inducible or repressible? Discuss.

6. Define an operon and describe the structure of the lac operon.

7. Explain the function of each part of the lac operon.

8. What are structural genes?

9. Explain the process of end-product inhibition as a gene regulation mechanism using a

named end-product.

10. Explain why glucose is regarded as a co-repressor in the function of the lac operon.

11. Differentiate between the lac operon in its induced state and in its repressed state.

4.6 Summary

Regulation of gene expression (or gene regulation) refers to the mechanisms which the cells use
to switch the processes of transcription and translation on and off. Gene regulation is important
as it allows an organism to express certain proteins only when they are needed in order to avoid
waste of materials and energy. The classical example of a gene regulation system is the lactose
operon (lac operon), discovered by Jacob and Monod, in which enzymes involved in lactose
metabolism are expressed by E. coli only in the presence of lactose and absence of glucose. An
operon is a group of genes which work together under the control of a single promoter. These
genes may be expressed (i.e. transcribed and translated) together and their protein products may
have related functions. Gene expression in eukaryotes is more complex than in prokaryotes and
its regulation takes place at different levels some of which are: (i) Transcription of DNA into
RNA(ii) Post transcriptional modification of RNA primary transcripts(iii)Translation of mRNA
into a polypeptide chain(iv) Post translational modification of the polypeptide chain(v) Cell
differentiation (vi) Growth and development. Each of these steps represents a potential point at
which the expression of eukaryotic genes may be turned on or off.
TOPIC 6: MUTAGENS AND GENE MUTATIONS

6.1 Introduction

Accurate DNA replication, transcription and translation all depend on the reliable pairing of
complementary bases. Errors which are called mutations occur, though very rarely, in all these
three processes. Mutations are heritable changes in genetic information. Gene mutations also
called point mutations are changes in the nucleotide sequence in a gene usually involving one
nucleotide. Gene mutations include substitutions, deletions and additions. Mutations can occur
spontaneously during replication or during cell division or they can be induced by various
environmental factors such as chemicals, radiation or even by biological agents mainly viral
infections. Mutagenesis is a process whereby a mutation is introduced into DNA through for
example physical factors (e.g. exposure of DNA to radiation) or chemical mutagens. Physical or
chemical mutagens can replace a base in the DNA, alter a base so that it specifically mispairs
with another base, or damage a base so that it can no longer pair with any base under normal
conditions.

6.2 Objectives: At the end of this topic you should be able to:
(i) List the various types of mutagens

(ii) Explain the effects of chemical, physical and biological mutagens

(iii) Describe point mutations - deletions, insertions, substitutions

(v) Describe and silent, neutral, missense, nonsense and frame shift mutation

(iv) Use sickle cell anaemia as an example of a point mutation.

6.3 Causes of gene mutations

Mutations are either spontaneous or induced. Spontaneous mutations occur naturally and
randomly while Induced mutations are caused by factors called mutagens.

6.3.1 Mutagens

The three common types of mutagens are (i) physical, (ii) chemical and (iii) biological.
Mutagens which cause cancer are also known as carcinogens.

6.3.1.1 Chemical mutagens

6.3.1.1.1 Deaminating agents

These mutagens remove amino groups from nitrogenous bases. For example they may remove
the amino group from cytosine thus converting it to uracil.

This may cause change in the base paring from CG through UA to AT as the affected DNA
undergoes replication. The overall change is: CG  UA  TA

Figure 6.1. Deamination: Nitrous acid (NA) deaminates cytosine to form uracil, which
bonds (pairs with adenine) like thymine. Source: Griffiths et al. 1994. An introduction to
Genetic Analysis Page 544.

Examples include nitrosamine, nitrous acid and sodium nitrite. Meat reddening is linked to the
use of sodium nitrite. Deaminating agents are linked to gastric cancer.

6.3.1.1.2 Alkylating agents

These mutagens add alkyl groups (e.g. CH3, CH2CH3) to nitrogenous bases. This may lead to
deletion of such bases especially the purines (A and G) or to mispairing of bases. Examples of
alkylating agents include dimethylsulphate, diethylsulphate and ethylmethane sulphate (EMS).
Figures 6.2. Alkylation: Ethylmethane sulphate (EMS) generates ethylation at the O-4
position of thymine. This causes the modified thymine to mispair with guanine leading to
TA  GC transition. Source: Griffiths et al. An introduction to Genetic Analysis. 1994. Page
543.
Ethylmethylsulphate (EMS) adds an ethyl group to the O-4 position of thymine. This causes the
modified thymine to O-4 ethylthymine which pairs with guanine. As the DNA replicate this
pairing leads to the change: TA  O-4Eth/G  GC.

Alkylating agents are linked to bladder, bronchial and blood cancer.

6.3.1.1.3 Base analogues

These mutagens are chemicals that look like normal bases and as such confuse the DNA
replication system. Examples are 5-bromouracil (5-BU), an analogue of thymine and 2-amino
purine (2-AP), an analogue of adenine.

Figure 6.3. Base analogues: 5 bromouracil (5BU) an analogue of thymine pairing with
adenine and 2-amino purine (2AP) an analogue of adenine pairing with cytosine. Source:
Griffiths et al. 1994. An introduction to Genetic Analysis. Pages 541-542.
These mutagens cause changes in base pairing. For example 5-BU may pair with guanine and
when the affected DNA replicates, it produces the change: TA  5BU/G  to GC. Similarly
2AP may pair with cytosine leading to the change: AT  2AP/C  GC.

6.3.1.1.4 Bulky addition products

These are huge molecules which are able to covalently bind to purines in DNA leading to the
deletion of such purines from the affected DNA. Aflatoxin B1 (AFB1) is a bulk addition mutagen
which is also a carcinogen found in stored seed crops such as groundnuts and maize. It is linked
to liver cancer and is correlated to Hepatitis B virus occurrence.

Figure 6.4. Bulky addition: The binding of Aflatoxin B1 to guanine. Source: Griffiths et al.
1994. An introduction to Genetic Analysis. Page 546.

For example the attachment of AFB1 to guanine will cause the deletion of the affected guanine
from the DNA leaving an apurinic site. The repair system usually inserts adenine in the site of
deletion. This leads to the change: CG  C*_  CA  TA

6.3.1.1.5 Intercalating agents

These molecules have three rings and resemble and mimic base pairs. They are able to insert
themselves (intercalate) between base pairs in the affected DNA double helix, thereby causing
frame shift mutations. Examples of intercalating agents include proflavin (which is used as a
topical antibacterial and urinary antiseptic), acridine orange (which is a dye used in the
laboratory detection of Mycobacterium) and ethidium bromide (which is used as a fluorescent
dye in agarose gel electrophoresis in PCR).
Figure 6.5. Intercalating agent: The structure of proflavin and the intercalation of
proflavin between the nitrogenous bases stacked at the centre of the DNA molecule. Source:
Griffiths et al. 1994. An introduction to Genetic Analysis. Page 544.

6.3.1.2 Physical mutagens

6.3.1.2.1 Ultraviolet (UV) radiation

UV generates pyrimidine (thymine) dimers which cause mutations and cancer (melanomas and
keratosis).

Figure 6. 6. Formation of a thymine dimer due to UV radiation. Source: Strickberger 1985.

Genetics. Page 491.

6.3.1.2.2 Ionization radiations

These include X-rays, gamma rays, neutrons and alpha rays. These radiations lead to the
generation of free radicals which cause breaks in the DNA. These breaks may result into
deletions and other types of mutations in the affected DNA.

6.3.1.3 Biological mutagens

6.3.1.3.1 Mutagenic viruses

Viruses are also potential mutagens. In instances of some acquired viral infections, the virus
attaches to the cell, transfer its genetic material into the cell thus altering the original gene,
causing mutation. Hepatitis B virus is an example of a DNA viral mutagen while Hepatitis C
virus is an example of an RNA virus. These viruses cause cancer in liver. Other examples of
viral mutagens are Human herpes virus, SV40 virus and Human Papilloma virus.

6.3.1.3.2 Transposable elements

A transposable element is a segment of DNA which is able to move from one position to another
in the same chromosome or from one chromososme to another. When it is inserted into a new
position on chromosomal DNA, it disrupt the function of the genes.

6.3.1.3.3 Mutagenic bacteria

Some bacteria such as Helicobacter pylori cause inflammation during which oxidative species
are produced, causing DNA damage by reducing efficiency of DNA repair systems thereby
increasing mutation.

6.4 Types of mutation

A mutation is a change in the amount, arrangement or structure of the DNA of an organism. This
change usually affects the phenotype and may be inherited by daughter cells from the mutant
mother cell.

Gene or point mutation is a mutation due to a change in one a gene. It is usually a change in a
single nucleotide base but may involve two or more bases. In this type of mutation new alleles of
a gene are produced and this change is then transcribed into RNA and finally translated into a
protein. Gene mutations may be spontaneous (natural) or may be induced (caused by external
agents called mutagens). The various types of gene mutations include; (i) addition or insertion,
(ii) deletion and (iii) substitution,

6.4.1 Insertions and deletions (Indels)

These are mutations which involve the insertion (addition) and deletion of bases in the DNA
sequence. Insertion or deletion of a single nucleotide or several nucleotides which are not
multiples of three will result into a frame shift in the genetic code. Frame shifts can lead to
missense or nonsense mutations and most of them have negative effects on the affected
individual.

6.4.2 Substitution

6.4.2.1 Transitional substitution

In such substitution, a purine is replaced by a purine or a pyrimidine replaced by a pyrimidine.

Transitional substitutions have very little or no effect on the type of protein synthesized.

6.4.2.2 Transverse substitution

In transverse substitution, a purine replaces a pyrimidine or vice-versa. Such a change will have
significant negative effects on the type of protein synthesized and in many cases the protein is
non-functional or inactive.

Figure 6.7. Types of possible base substitutions. The symbol α represents transitional
substitutions while β represents transerversional substitutions.
6.4.2.2.1 Sickle cell anaemia

One common example of transverse substitution in humans is sickle cell haemoglobin (HbS)
which causes a disease called sickle cell anaemia. The normal haemoglobin (Hb A) has glutamate
at position 6 of the beta-heamoglobin chain while HbS has valine at the same position. This
difference is due to a change from the codon GAA (for glutamate) to the codon GUA (for
valine). Glutamate is hydrophilic and is able to stick out into the aqueous medium of the blood
allowing the red blood cells to have their normal shape. Valine on the other hand is hydrophobic
and tends to stick away from the aqueous medium of the blood leading to the sickle shape in the
folding of the red blood cells.

Table 6.1. Illustration of point mutations

Type of mutation Triplet code

Wild type ACC CAC UCU GGA AUU GAA GCA
Thr His ser Gly ile glu Ala
Same silent/null mutation ACC CAU UCU GGA AUU GAA GCA
Thr His ser Gly ile glu Ala
Missense mutation ACC CAC UCU GGA AUU GUA GCA
Thr His ser Gly ile val Ala
Neutral mutation ACC CAC UCU GGA GUU GAA GCA
Thr His ser Gly val glu Ala
Nonsense mutation ACC CAC UCU UGA AUU GAA GCA
Thr His ser Stop --- --- ---
Negative (-1) Frame shift mutation ACC ACU CUG GAA UUG AAG CA
Deletion of C at position 4 Thr Thr leu Glu
Positive (+1) Frame shift mutation ACC CAA CUC UGG AAU UGA AGC
Insertion of A at position 6 Thr gln leu Trp Ile stop ---

(i) Silent or null mutation is when the same amino acid is coded for; e.g. if CAC is changed to
CAU, histidine will still be translated.

(ii) A neutral mutation is where the protein will not change much because the amino acids coded
for are functionally the same. If AUU (hydrophobic isoleucine) is replaced by GUU
(hydrophobic valine).

(iii) A missense mutation causes the substitution of one amino acid for another functionally
different amino acid; e.g. when GAA (hydrophilic glutamate) changes to GUA (hydrophobic
valine).
(iv) Nonsense mutations occur when a codon mutates into UGA, UAA or UAG stop codons.

(v) Frame shift mutations occur when the reading frame on the mRNA is disturbed. They are
caused by the deletion (negative frame shift) or insertion (positive frame shift) of bases in the
DNA.

6.5 Revision questions

1. Define a base analogue. Use examples to explain how base analogues induce mutations in
DNA.
2. Explain specific mispairing as it applies to gene mutations.
3. Using a named alkylating agent, describe the mechanism used by these mutagenic
agents to induce mutations.
4. (a) Explain how a mutagenic virus induces mutations in the host DNA.
(b) Give examples of some mutagenic viruses.
5. (a) Name the various groups of chemical mutagens.
(b) Explain how each of these groups of mutagens induce changes in the affected
DNA.
6. Compare and contrast a mutagen and a carcinogen.
7. Illustrate the three types of point mutations.
8. Using examples, describe the following types of gene mutation:
(a) Missense mutation (b) Neutral mutation (c) Nonsense mutation
9. Name the type of point mutation which causes sickle cell anaemia and explain how
this happens and the effect of sickle cell anaemia on the red blood cell.
10. Differentiate between spontaneous mutations and induced mutations.

6.6 Summary

Mutations result in abnormal function of cells. They may be spontaneous or due to factors called
mutagens. There are various types of mutagens such as chemical, physical and biological. Gene
or point mutations are of various types and include deletions, insertions and substitution. The
gene mutations are described according to the effect on the function of the final polypeptide
product and include silent, neutral, missense, nonsense and frame shift mutations. Sickle cell
anaemia as an example of a serious disease caused by a point mutation.
TOPIC 6 CHROMOSOMAL THEORY OF INHERITANCE

6.1 Introduction

The chromosomal theory has revolutionized the way many scientists perceive cell division and
cell reproduction. This theory was developed over a number of decades has led to many
developments in cell biology, genetics, molecular biology which have applications in agriculture
and medicine among other applications.

6.2 Objectives: At the end of this topic you should be able to:
1. Outline the series of events which led to the discovery of chromosomes

2. Link Chromosome behaviour to inheritance of genetic characteristics

3. Describe the molecular structure of chromosomes.

6.3 Discovery of Chromosomes

The build up to the discovery of chromosomes started with Gregor Mendel, an Austrian Priest
and researcher, who proposed two laws of heredity. Meanwhile, Charles Darwin an English
r1esearcher postulated that ‘gemmules’ traveled from every body part to the sexual organs,
where they were stored. Walther Flemming a German biologist discovered the presence of
threadlike structures in the cell nucleus which were able to absorb basic dyes and became
coloured. These were called chromatin or chromosomes (coloured body). He also studied the
behaviour of the chromosomes in cells undergoing cell division (mitosis). Theodor Boveri, a
German embryologist discovered that the number of chromosomes was reduced in the gametes
during meiosis. Walter Sutton, an American scientist observed that the behavior of chromosome
undergoing meiosis during gamete formation was consistent (in agreement) with Mendel's
second law of heredity. "I may finally call attention to the probability that the association of
paternal and maternal chromosomes in pairs and their subsequent separation during the reducing
division as indicated above may constitute the physical basis of the Mendelian law of heredity."
Sutton, 2002. Thomas Morgan, an American scientist together with his colleagues (who included
his wife) provided physical evidence to link behaviour of chromosomes to an inherited
characteristic. Their experiments showed very clearly that the rare white color of the eyes in
Drosophila was due to a mutation of a gene responsible for eye color which is located on the X
chromosome of Drosophila.

6.4 Nature of Chromosomes

In prokaryotes, genes are located in the cytoplasm either on genomic (chromosomal) DNA or on
extra chromosomal (plasmid) DNA. In Eukaryotes genes are either located in the nucleus on
chromosomal DNA or in the cytoplasm on mitochondrial and/or chloroplast DNA.

6.4.1 Relationship between chromosome and DNA

Chromosomes are the carriers of the genes and represent the material basis of inheritance. Chemical
analysis shows that chromosomes carry DNA which is the genetic material. DNA stores and transmits
genetic information from one generation to another. It remains constant in a given species and is
a large, stable macromolecule which is not metabolised. Prokaryotic (bacterial) chromosomes
are made up of naked double-stranded DNA located in their cytoplasm. In most eukaryotes the
nucleus of each cell contains several pairs of homologous chromosomes which contain DNA
tightly bound to proteins.

6.5 Eukaryotic chromosome structure

Eukaryotic chromosomes are made up of equal amounts of DNA and proteins (basic histones and
acidic non-histones). There are many kinds of acidic proteins but only five basic histones (H1,
H2A, H2B, H3 and H4) are common to most species. The ‘beads’ seen when chromosomes are
observed under a microscope during interphase are the nucleosomes which consist of an octamer
(group of 8 molecules) comprising two molecules each of histones H2A, H2B, H3 and H4 around
which DNA is wrapped (11/3 turns). Histone 1 is involved in the packaging of nucleosomes into
the solenoid fibre (Figure 6.1).

Figure 6.1. The arrangement of DNA in the chromosome. Source: Jones and Karp. 1994.
Introducing Genetics. Pages 181-183.

The large amount of DNA in eukaryotic chromosomes creates a problem of packaging and
organization. The chromosomes must allow for gene activity and must be able to replicate during
cell division (mitosis and meiosis). The chromosomes are in the extended position during the
resting phase and DNA replication but are tightly packed and shortened during cell division.
When the chromosome is loosely coiled, during interphase, the turns that the DNA makes about
the nucleosome (octamer) give a packing ratio of 10:1 and further coiling into the solenoid fibre
gives a ratio of 100:1 is achieved. When the chromatin is further condensed by super coiling and
folding at metaphase, the packing ratio increases up to 10 000:1.

6.6 Genes and alleles

6.6.1 The gene: A gene is a sequence of nucleotide pairs along a DNA molecule, which codes
for a ribonucleic acid (RNA) or a polypeptide product. Genes which code for polypeptides are
divided into two main classes. These are: (1) structural genes, which code for functional proteins
such as enzymes, hormones, membrane proteins, antibodies, storage proteins, etc., and (2)
regulatory genes, which serve to control or regulate the activity of other genes

6.6.2 The allele: In diploid organisms the chromosomes exist in homologous pairs with one set
coming from the mother and the other from the father. Each member of a homologous pair
carries one of the alleles of each character (gene) at corresponding positions called loci (sing.
locus) along its length.

An allele is a particular form of a gene occupying a fixed locus (place) on a chromosome.

Most genes have two alleles (allelic forms).

6.7 Revision questions

1. Name some scientists who contributed towards the discovery of chromosomes.

2. Describe the structure of a eukaryotic chromosome.

3. Distinguish between a gene and an allele.

6.8 Summary

The chromosome theory of inheritance was the collaborative result of multiple researchers working
over many years. The first ideas were started 1860s, when Gregor Mendel and Charles Darwin
each proposed possible systems of heredity. Later on, Walther Flemming discovered
chromosomes and described their behaviour during mitosis. The idea of a connection between
chromosomes and heredity was strengthened by research done by Theodor Boveri and Walter
Sutton, but direct evidence in support of chromosome theory came from Thomas Hunt Morgan's
experiments with fruit flies (Drosophila). Therefore, after nearly 50 years of speculation,
scientists were finally able to confirm that chromosomes were indeed the physical carriers of
hereditary information.
7. CELL CYCLE

7.1 Introduction
The cell cycle is the series of events that take place in a cell leading to its division and
duplication (replication). It is the period from the beginning of one cell division to the beginning
of the next cell division. The cell may undergo mitotic division or meiotic division depending on
the cell type.

7.2 Objectives : At the end of this topic you should be able to:
(i) Identify the stages in the cell cycle and describe the main events of each stage

(ii) Describe the various stages of mitosis emphasizing the behaviour of chromosomes

(iii) Describe the various stages of meiosis emphasizing the behaviour of chromosomes

(iv) Explain the significance of mitosis

(v) Explain the significance of meiosis

(vi) Contrast between the events of mitosis and meiosis

7.3 Stages of the Cell cycle

The time it takes to complete one cycle is called the generation time and it varies according to
the type of cell involved and according to environment of the cells. In eukaryotes, the cell cycle
can be divided in two periods: (i) interphase during which the cell grows by synthesizing
molecules needed for duplicating its DNA, (ii) the mitotic (M) phase during which the cell splits
into two separate "daughter cells”. The time it takes to complete one cycle is called the
generation time and it varies according to the type of cell involved and according to environment
of the cells. In eukaryotes, the cell cycle can be divided in two periods: (i) interphase during
which the cell grows by synthesizing molecules needed for duplicating its DNA, (ii) the mitotic
(M) phase during which the cell splits into two separate "daughter cells".
Figure 7.1 Diagram of a typical cell reproductive cycle. Source: https://en.wikipedia.org
/wiki/Cell cycle.

Cell division involves two major processes; karyokinesis (division of the nucleus) and
cytokinesis (division of the cytoplasm). In some cases a cell may only undergo nuclear division
without cytokinesis resulting in a cell with two or more nuclei as seen in muscle cells. The
lengths of these phases differ from one cell type to another. Normal mammalian cells growing in
tissue culture, for example, usually require 18-24 hours at 37°C to complete their cell cycle.

7.3.1 Interphase

Interphase, also known as the preparatory phase, takes place before mitosis and cytokinesis.
Before a cell can enter cell division, it needs to synthesize different molecules. All of the
preparations are done during the interphase which is in three stages; (i) G1, (ii) S, and (iii) G2.
During interphase, the nucleus and cytoplasm do not divide but the cell merely prepares for
division.

7.3.2 Gap 1 phase- G1

G1 is the growth phase and is the first part of interphase, from the end of the previous M phase
until the beginning of DNA synthesis. During this phase the biosynthetic activities of the cell
take place at a high rate. This phase uses the 20 amino acids to form millions of enzymes and
other proteins that are required in the S phase for DNA replication. The duration of G1 is highly
variable but is relatively long.

7.3.3 Synthesis phase - S

The S phase starts with DNA replication and when it is complete, all of the chromosomes have
been replicated. This means each chromosome will have two sister chromatids. Thus, during this
phase, the amount of DNA in the cell is doubled. This phase is completed very quickly to avoid
damage to the exposed base nucleotides which are sensitive to mutagens. DNA synthesis starts
at several positions on each chromosome thereby reducing the time required to replicate the
whole chromosome.

7.3.4 Gap 2 phase - G2

This is also called the post- DNA synthesis phase. It is the gap between DNA synthesis and
mitosis during which the cell will continue to grow. The cell makes sure that everything is ready
for it to enter the M (mitosis) phase.

7.4 Mitosis

The period of the cell cycle when the cell is undergoing division is called the mitotic phase (M
phase). It is the process by which the chromosomes of the cell nucleus a cell separate into two
identical sets and end up in two separate nuclei. It is a form of karyokinesis (nuclear division)
which is followed by cytokinesis (cell division) in which the nucleus and cytoplasm, organelles,
and cell membrane are divided into roughly equal amounts. The process of mitosis is fast and
quite complex. It is divided into (i) prophase, (ii) metaphase, (iii) anaphase and (iv) telophase.
The times spent in each of these phases are quite different but prophase usually requires longer
durations than the other phases and metaphase is the shortest.

Mitosis is important for the maintenance of the chromosomal set; each daughter cell receives
chromosomes that are identical in composition and equal in number to the chromosomes of the
mother cell.

Mitosis is important for the following functions:

(i) Growth and development

(ii) Cell replacement and tissue repair e.g. in the skin and digestive and red blood cells
(iii) Tissue and organ regeneration: e.g , starfish regenerate lost arms through mitosis
(iv) Asexual reproduction e.g budding in yeast and hydra and vegetative propagation in plants.
PROPHASE METAPHASE

The chromatin is condensing The chromatids align at the

into chromosomes. metaphase plate.

ANAPHASE TELOPHASE

The chromosomes split and the The decondensing chromosomes are surrounded by
kinetochore microtubules shorten nuclear membranes. Cytokinesis has already begun;
the pinched area is known as the cleavage furrow.
Figure 7.2. Diagrams of mitosis in a hypothetical cell. Source: https://www.google.com/
search?q= mitosis&biw=1440&bih=789&tbm=isch&tbo= u&source=univ&sa= X&sqi=
2&ved=0ahUKEwiR__LOvq3JAhWBPxoKHTwTDMMQsAQIVA#imgrc=g0yz1VgoLnpHyM
%3A.

7.4.1 Prophase: Chromosomes become condensed by coiling and thickening. The chromosomes
have replicated into chromatids but are still attached at the centromeres. The nuclear membrane
disappears.

7.4.2 Metaphase: Chromatids align at the metaphase plate (equatorial region) and are still
attached at the centromeres.

7.4.3 Anaphase: Chromatids split into separate chromosomes and the two sets migrate to the
opposite poles of the cell.
7.4.4 Telophase: Chromosomes uncoil and become thin. The nuclear membrane reappears and
each set of chromosomes is enclosed by a separate nuclear membrane. Cytokinesis has already
begun.

7.4.5 Cytokinesis: In animals, the cell separates into two through a pinched area known as the
cleavage furrow while in plants the cells separates into two by the cell plate. At the end of
mitosis two daughter cells are produced and each of them contains the same number of
chromosomes as the mother cell.

7.4.6 Role of centrioles and microtubules (spindle fibres): Microtubules (spindle fibres) which
found in both plant and animal cells hold the chromosomes in place and also help with the
separation of the chromatids into chromosomes. Centrioles which are present in animal cells but
not in plant cells help to organize the spindle fibres during cell division.

7.4.7 Rapidly dividing cells: In humans, examples of rapidly dividing cells are those in the
epithelium of the digestive track, in the skin and the stem cells that are used to produce blood
cells. The rate at which cells divide is genetically controlled to prevent abnormal division,
apoptosis and cancerous tumours.

7.5. Meiosis

Meiosis is a type of cell division which is involved in the production of gametes that are
involved in sexual reproduction. The male gametes are generally called sperm and the female
gametes are generally called eggs. Meiosis is not a cycle like mitosis because the end products
(gametes) must first undergo fertilization to produce a zygote which develops into an individual
organism. This individual will then produce gametes which will undergo the same fate. Meiosis
occurs only in the specialized cells (germ line) of the reproductive organs (gonads). In animals,
the testes are male gonads and the ovaries are female gonads. Gametes contain the haploid
number (n) of chromosomes, but originate from diploid (2n) cells of the germ line. Meiosis
ensures that the number of chromosomes is reduced by half during gamete formation in order to
maintain the chromosome number of the species after fertilization. Meiosis involves a single
DNA/chromosome replication and two divisions of the cytoplasm (meiosis I and meiosis II).
The DNA/chromosome replication takes place during interphase before meiosis I. In the end, one
diploid mother cell divides into four haploid daughter cells as a consequence of meiosis I and
meiosis II.
7.5.1 Meiosis I
The first meiotic division is a reductional division that produces two haploid cells from a single
diploid cell. Meiosis I consists of four major phases (i) prophase I, (ii) metaphase I, (iii)
anaphase I and (iv) telophase I.
7.5.1.1 Prophase I
Prophase I differs from the prophase of mitosis in that homologous chromosomes come to lie
side by side in a pairing process called synapsis. Each pair of chromosomes is called a bivalent
(2 chromosomes) with each chromosome consisting of two identical sister chromatids. A
bivalent may also be called a tetrad (4 chromatids). During synapsis, non-sister chromatids (one
from each of the paired chromosomes) of a tetrad may cross over and exchange portions. The
point of exchange is called a chiasma (plural = chiasmata). Therefore, at a given chiasma, only
two of the four chromatids cross over in a random manner. Generally, the longer the
chromosome, the higher the number of crossovers.

7.5.1.2 Metaphase I
During metaphase I, the bivalents align themselves at random on the equatorial plane. This
random orientation promotes independent assortment of the chromosomes and their genes.
7.5.1.3 Anaphase I
During anaphase I, the centromeres do not divide, but continue to hold sister chromatids
together. Because of crossovers, sister chromatids may no longer be genetically identical.
Homologous chromosomes (each consisting of 2 sister chromatids) separate and move to
opposite poles. This movement reduces the chromosome number from the diploid (2n) condition
to the haploid (n) condition.
7.5.1.4 Telophase I
The first meiotic division effectively ends when the chromosomes arrive at the poles. Each
daughter cell now has half the number of chromosomes but each chromosome consists of a pair
of chromatids. The nuclear membrane reappears and surrounds the haploid set of chromosomes.
The chromosomes uncoil back into chromatin.
Cytokinesis in telophase I divides the diploid mother cell into 2 haploid daughter cells. This
involves the formation of a cleavage furrow in animal cells or the formation of the cell plate in
plant cells, occurs. Sister chromatids remain attached during telophase I.
7.5.1.5 Interkinesis
The period between the first and second meiotic divisions is called interkinesis or interphase II.
The DNA does not replicate during interkinesis.
7.5.2 Meiosis II
The second meiotic division (meiosis II) is an equational division (mitosis-like), in which sister
chromatids of the haploid cells are separated). This process is similar to mitosis, though the cells
produced have half the number of chromosomes. Meiosis II also consists of four major phases
(prophase II. metaphase II, anaphase II, and telophase II).

7.5.2.1 Prophase II
In prophase II, the nuclear membrane disappears and the spindle fibres reappear. The chromatids
shorten and thicken in readiness for the second meiotic division.
7.5.2.2 Metaphase II
At metaphase II, the individual chromosomes line up on the equatorial plane. The new equatorial
metaphase plate is rotated by 90 degrees compared to meiosis I plate and is therefore
perpendicular to the metaphase I plate.
7.5.2.3Anaphase II
During anaphase II, the centromeres of each chromosome divide, allowing the sister chromatids
to be pulled apart in an equal division (mitosis-like) by the spindle fibres. The sister chromatids
which are now called chromosomes move toward opposing poles.
7.5.2.4 Telophase II
During telophase II, which is similar to telophase I, the chromosomes uncoil and lengthen. The
spindle fibres disappear and the nuclear membrane reforms and there is formation of either a
cleavage furrow (in animal cells) or cell plate (in plants) producing a total of four daughter cells,
each with a haploid set (half the number) of chromosomes.
Figure 7.3. Diagrams of meiosis in a hypothetical cell. Source: https://www.google.com/search?
q=meiosis&tbm=isch&tbo=u&source=univ&sa=X&ved=0ahUKEwjwssuHz63JAhXIVhoKHcK
iBXcQsAQISA&biw=1440&bih=789.
7.5 Revision questions

1. Name the stages of the cell cycle and briefly describe the major events of each stage.
2. Explain the function(s) of mitosis.
3. Explain the function (s) of meiosis.
4. Name the stages of mitosis and briefly describe the major events of each stage.
5. Name the stages of meiosis and briefly describe the major events of each stage.
6. Human cells normally have 46 chromosomes. For each of the following stages, state
the number of chromosomes present in a human cell:
a. Metaphase of mitosis
b. Metaphase I of meiosis
c. Telophase of mitosis
d. Telophase I of meiosis
e. Telophase II of meiosis
(In your answer, count sister chromatids as 1 chromosome).
7. Four of the following events are part of both meiosis and mitosis but only one is
meiotic. Identify it.
a. Chromatid formation
b. Spindle formation
c. Chromosome condensation
d. Chromosome movement to poles
e. Chromosome pairing
8. Define the following terms:
(a) Diploid (b) Haploid (c) Homologous chromosomes
9. Distinguish between chromatin, chromatid and chromosome
10. List the major differences between mitosis and meiosis

7.6 Summary

The cell cycle is the period from the beginning of one cell division to the beginning of the next
division. During interphase, the cell grows and prepares for the next division. The cell also
duplicates its DNA during the S phase of interphase. The division of the cell takes place during
the M phase. All somatic cells in a multicellular organism are descendant of one original cell, the
fertilized egg or zygote, through a divisional process called mitosis. During mitosis, a complete,
identical set of chromosomes is distributed to each daughter cell. It consists of prophase,
metaphase, anaphase and telophase. Sexual reproduction involves the production of gametes
(gametogenesis) and the union of a haploid male and a haploid female gamete (fertilization) to
produce a diploid zygote. Male gametes are sperms or pollen and female gametes are eggs (ova),
or ovules. Gametogenesis occurs only in the specialized cells (germ line) of the reproductive
organs (gonads) of the mature individual. Meiosis reduces the chromosome number by half in
the daughter cells as compared to the mother cell. It is required in sexually reproducing
organisms because it results in gametes which have a haploid number of chromosomes and after
fertilization the zygote has the diploid number.
TOPIC 8 CLASSICAL MENDELIAN GENETICS

8.1 Introduction

Genetics is defined as the study of variation and heredity. It is concerned with the origin of
variation and the maintenance as well as inheritance of this variation in from generation to
generation. Gregor Mendel is considered the father of genetics. Between 1856 and 1863 Mendel
cultivated and tested almost 28,000 pea plants. His knowledge of mathematics enabled him to
understand the F2 ratios and to see that they were a result of randomly combining pairs of
cellular factors. He linked the distribution of the pair of factors to the expansion of the binomial
expression (A+a) (A+a) or (A+a)2. Mendel published the results from his studies in 1866 in
which he proposed two laws of inheritance in sexually reproducing organisms. He proposed the
existence of a pair of factors controlling each character transmitted from parents to offspring. He
therefore laid ground for the particulate theory of inheritance. However, no one paid attention to
Mendel’s results until the early 1900s. Genetics can be applied in medicine, pharmaceutical
industry, forensics, conservation, etc.

8.2 Objectives: At the end of this topic you should be able to

1. Define genetics and explain its significance.

2. Explain the effect of the environment on gene expression.
3. Explain and apply Mendel’s First Law of Segregation and of the monohybrid cross.
4. Explain and apply knowledge of the dihybrid cross and Mendel’s Second Law of Independent
Assortment.
5. Use the Punnet square or probabilities to find the phenotypic ratios in the F2 of a dihybrid
cross involving two characters determined by two independent genes.
6. Explain the principle behind the Chi-squared test (χ2 Test) and apply it to solve genetic
problems.

8.3 Common genetic terms

8.3.1 The genotype: This is the genetic constitution (make-up) of an individual. It shows the
types of alleles present in the individual. E.g. TT is the genotype for homozygous tall pea plants,
Tt = heterozygous tall, tt = homozygous short.
8.3.2 The phenotype: This is the physical appearance or function of an organism as a result of
its genotype and its environment. The phenotype results from the expression of the genetic
information through the protein (polypeptide) product. E.g. the two phenotypes for height in pea
plants are tall and short.

8.3.3 Dominant allele

A dominant allele is an allele that masks (hides) the expression of another allele of the same
gene. A dominant allele produces a dominant phenotypic character. E.g. the allele (T) for tall
height in pea plants is dominant to the allele (t) for short height (T >t). TT and Tt plants will both
have the tall phenotype.

8.3.4 Codominant alleles

Codominant alleles are contrasting alleles which are both expressed in a heterozygote (F1). The
heterozygote with such alleles exhibits the relevant characteristics of both parents. For example,
coat colour of cattle is determined by two alleles R (red) and W (white). When red (RR) and
white (WW) cattle are crossed, they produce roan (RW) offspring, which possess both
red and white hairs on their skin;

8.3.5 Incompletely dominant alleles

A heterozygote with incompletely dominant alleles will exhibit an intermediate between the two
homozygous parental phenotypic characters. E.g. (i) The flower colour in the four o’ clock plant
(Mirabilis jalapa) in which the F2 generation has three genotypes; RR (red), RW (pink) and WW
(white).
8.3.6 Recessive alleles
A recessive allele is one whose effect is masked (hidden) by the presence of a dominant allele of
the same gene. A recessive character is only expressed by the recessive alleles in their
homozygote state. E.g. (i) The allele (t) for short height in the pea plant is recessive to the allele
for tall height. Plants with genotype tt will be short, TT and Tt plants will be tall (T > t).

8.4 Environmental effects on gene expression

The expression of certain genes is influenced by the environment in which they are expressed.
The environmental may enhance or inhibit the expression of certain genes. For example, height
in humans is influenced by the diet and general health of the individual. Studies using identical
twins have shown that although the twins carry the same gene, their phenotypes can be different
if they are raised in different environments.

8.5 Mendel’s experiments

Gregor Mendel observed that certain characters of the garden pea plant had two alternative
forms. He saw that some plants bred true for one character while the other plants bred true for
the alternative character. In his study, Mendel used the seven characters of garden peas as shown
in Table 8.1.

Table 8.1 Characters and their alternative forms in the garden peas used by Mendel

The seven characters chosen by Mendel are constant and easily recognizable. For example, the
flowers are clearly visible and the parts are easily distinguishable. The characters have
contrasting traits (forms) e.g. smooth vs wrinkled seeds. The hybrids are fertile and both cross
and self-fertilization are possible. Self-pollination is naturally favoured because the reproductive
parts of the flowers are covered by the keel (two petals) and only opens after pollination has been
completed (cleistogamy). Cross-pollination is possible by emasculation which involves the
opening of the young bud of the female parent plant, removal of the keel and stamen using
forceps and dusting the stigma with pollen from a specific male parent plant. The pea seeds are
cheap, readily available, take up little space, have a short generation time and produce many
offspring.

To study the inheritance of stem height, Mendel crossed tall plants with short plants and got
hybrid plants which were all tall. He then self-pollinated the hybrid tall plants among themselves
and noticed that they produced a mixture of tall and short plants. He also used the other
characters and got similar results. In other experiments he studied the simultaneous inheritance
of two characters (such as seed shape and seed colour) and observed that the two forms of each
character are inherited independently of each other. From this work, Mendel was able to
understand the nature of inheritance and came up with his two principles or laws of inheritance
i.e. (1) The Law of Segregation and (2) The Law of Independent Assortment.

8.6. Mendel’s Laws of Heredity

8.6.1 Mendel’s First Law - The Law of Segregation

This Law states that during gamete formation, contrasting forms of a gene separate in equal
numbers. This law is best illustrated by a monohybrid cross.

8.6.1.1 Monohybrid cross

This is a cross between homozygous parents that differ only in one pair of alleles of one gene,
controlling one character. For example, stem height in garden peas is controlled by one gene in
which a pure breeding tall plant has genotype TT and a pure breeding short plant has a genotype
tt. Mendel carried out separate monohybrid crosses for each of the seven characters in his study
of their inheritance and he got similar results.
8.6.1.2 F1 hybrids

The progeny or offspring of a monohybrid cross is called the first filial generation or F1 hybrid.
Usually reciprocal crosses are made in a monohybrid cross. For example, pollen from tall plants
can be used to fertilise the ovules of the short plants in one cross while pollen from short plants
can be used to fertilise the ovules of tall plant in the other cross.

Figure 8.1 A reciprocal cross

Mendel observed that when seeds from the F1 plants were grown, they were all identical: they all
resembled the tall plants with no short or intermediate forms. Mendel reasoned that both the tall
and short forms of the character were present in the hybrid but only the tall form was expressed.
He called the tall form dominant and the short form which did not appear, he called recessive.
He noted that tall was dominant in both cases of the reciprocal cross (Figure 8.1).

8.6.1.3 The F2 generation

The second filial or F2 generation is raised by allowing the F1 hybrids to self-pollinate. When
Mendel examined the F2 plants, the recessive (short) character reappeared and was seen together
with the dominant (tall character). The numbers of the plants were in the ratio of 3 tall: 1 short.

The phenotypes of parental, F1 and F2 generations are summarised in Figure 8.2 below.

Figure 8.2. Parental, F1 and F2 generations

Mendel interpreted that the 3:1 phenotypic ratio represented a 1: 2: 1 genotypic ratio representing
the binomial expression: (T + t) (T + t) = 1TT + 2Tt + 1tt

This ratio gave Mendel the idea that the character that is passed on during reproduction existed in
two alternative forms (T and t) which are able to combine together randomly in pairs. The
formation of gametes, segregation of the forms (alleles) and production of F1 and F2 generations
are illustrated in Figure 8.3.

Figure 8.3. Segregation of alleles in a monohybrid cross

8.6.1.4 The Punnet square

R.C. Punnet devised the Punnet Square method which clearly shows how the alternative forms
of a character combine to produce the F2 progeny.

Figure 8.4 F2 progeny is depicted by the 2x2 Punnet square (Table 8.2) of a monohybrid cross

Table 8.2 Punnet square of the F2 generation of a monohybrid cross

Phenotypic ratio = 3T_ : 1tt and Genotypic ratio = 1TT: 2Tt: 1tt

8.6.1.5 Testcross

This is a breeding test, in which dominant phenotype (F1 hybrid) is crossed with the recessive
homozygote in order to verify whether it is an F1 hybrid (heterozygote) or a homozygous
dominant genotype. The results of a testcross confirm that there are two kinds of alleles in the
hybrid. A testcross in which an individual is crossed with one of its own parents is referred to as
a backcross.
Testcross results (i)

Figure 8.5. Test cross (i) results

Testcross results (ii)

Figure 8.6. Test cross (ii) results

When ½ of the progeny of the testcross are tall and ½ are short, then the dominant genotype is
confirmed to be heterozygous (F1 hybrid). When all the progeny of the test cross are tall then the
dominant genotype is confirmed to be homozygous.
8.6.2 Mendel’s 2nd Law – The Law of Independent Assortment

This law states that ‘When two or more unlinked pairs genes are brought together in a cross, their
alleles assort (segregate) independently of each other as a result of meiosis’. This law is best
illustrated by a dihybrid cross.

8.6.2.1 Dihybrid cross

Figure 8.7. A dihybrid cross showing independent segregation (assortment).

The formation of the F2 generation can be presented in a Punnet square as in Table 8.3.
Table 8.3 Punnet square of the F2 generation of a dihybrid cross

The genotypes of the F2 of a dihybrid cross do not show any pattern while the phenotypes show a
regular pattern.

There are four possible phenotypes in the F2 i.e. (i) Round yellow - Parental (ii) round green –
Parental (iii) Wrinkled yellow – Recombinant and (iv) wrinkled green – Recombinant.

The phenotypic ratio is:

9
/16 round yellow: 3/16 round green: 3/16 wrinkled yellow: 3/16 wrinkled green = 9:3:3.1

9
/16 R_Y_ : 3/16 R_yy : 3/16 rrY_ : 1/16 rryy

8.6.2.2 Dihybrid inheritance test cross

This is a cross between a double dominant phenotype and a double recessive phenotype which
confirms whether the double dominant phenotype is homozygous or heterozygous. A reciprocal
cross is recommended.
Figure 8.8. A dihybrid test cross

8.7. The Chi-squared test

The Chi-squared test (χ2 Test) is a statistical test used to confirm whether the differences
between the observed values and the expected values are significant or not. It tests whether
deviations in the observed ratios from the expected Mendelian ratios of a given cross are due to
chance or due to real departure (difference). The number of observations used in the chi-squared
test must be based on 30 individuals or more in order to get reliable results.
Mendel had to work with very large numbers of plants to get reliable results because there were
no statistical methods for assessing the reliability of his data available during his time.
In deciding whether the results obtained are within the expected ratio, the calculated χ2 value is
compared with the tabulated χ2 value. If the calculated value is greater than the tabulated (critical
value), then the departure from the expected ratio is significant, i.e. there is real departure from
the expected ration; and vice-versa.
The formula for calculating the chi-squared is:

Where Σ = sum of; O = observed values; E = expected values.

The tabulated χ2 value is obtained from the χ2 statistical table at 5% probability and at the
appropriate degrees of freedom (df), where df = number of classes – 1.

8.7.1 Worked example

Consider the progeny from a cross between two red-flowered F1 plants as follows:
Rr x Rr
R is the dominant allele which determines red flowers in Rr and RR plants
r is the recessive allele which gives white flowers only in rr plants.
The results from 200 F2 plants are 148 red flowered plants and 52 white flowered plants. Use the
chi-squared test to determine whether the two results are in the expected Mendelian ratio of an F2
progeny of a monohybrid cross or not.
Solution
Under perfect conditions, we expect a 3 red: 1white phenotypic ratio in the F2 progeny i.e. 150
red and 50 white flowered plants for the sample of 200. In real situations the conditions are not
perfect and therefore a chi-squared test should be used.
Chi-squared test of results (i) of 152 red and 48 white

Character O E d d2 d2/E

Red 148 150 -2 4 0.02667

White 52 50 2 4 0.08

Total (Σ) 200 200 0 0.10667

The calculated χ2 value is 0.11

There are two classes (red and white) hence one degree of freedom
(df) = number of classes – 1 = 2 – 1 = 1.
From the χ2 table, the value for 1 degree of freedom at 5% probability = 3.841 (highlighted in the
Table 8.4).
Table 8. 4: An extract from the Chi-squared table

In conclusion the calculated χ2 value of 0.11 is less than the table χ2 value of 3.841. Therefore
the calculated χ2 value is not significant and the observed values agree with the expected values
meaning that the results are according to the expected Mendelian phenotypic ratio of 3:1.

Table 8.5 Expected ratios for monohybrid and dihybrid crosses

8.8 Revision exercise

1. Name the founder of genetics and state his major findings summarized in his two laws.
2. Explain the relationship between a chromosome, a gene and an allele.
3. Explain the chromosomal theory of inheritance.
4. Define the terms gene, allele, genotype, phenotype, homozygous, heterozygous,
homologous and non-homologous.
5. Differentiate between the following pairs of terms: homologous/diploid
dominant/recessive; allele/gene; F1/F2, homozygous/heterozygous;
genotype/phenotype; monohybrid/dihybrid.
6. Define dominant, recessive, codominant, incompletely dominant and lethal alleles.
7. State the difference between the number of alleles in a somatic cell with that in a
gametic cell.
8. Name two major factors which determine the phenotype of an individual.
9. Comment on the randomness of the fusion of given a sperm and a given ovum.
10. Consider the progeny from a cross between two red-flowered F1 plants as follows:
Rr x Rr
R is the dominant allele which determines red flowers in Rr and RR plants
r is the recessive allele which gives white flowers only in rr plants.
The results from 200 F2 plants are 165 red flowered plants and 35 white flowered plants. Use the
chi-squared test to determine whether the two results are in the expected Mendelian ratio of an F2
progeny of a monohybrid cross or not.
11. If two heterozygous organisms are crossed, and the trait being studied displays
complete dominance, one may expect the following type of phenotypic ratio in the
offspring: (a) 3:1 (b) 1:2:2 (c) 9:3:3:1 (d) the offspring are all alike (no ratio).
12. When red-flowered and white flowered four-0’clocks are crossed, the pink flowered
offspring exhibit: (a) complete dominance (b) lethality (c) incomplete dominance (d)
codominance
13. State the number of alleles of the gene for pea-colour that a pea plant possess in
(a) the cells of the pea coat
(b) the cells of the stem
(c) the cells of the root
(d) the gametes

14. Indicate which of the alleles in the following example are dominant, codominant or
incompletely dominant:
(a) a chicken with the allele for black feathers and the allele for white feathers possesses
blue feathers.
(b) A cat with the allele for black coat and the allele for red coat shows a patched coat of
black and red.
(c) A chicken with the allele for straight feathers and the allele for frizzled feathers has
mildly frizzled feathers.
(d) A person with the allele for blood group M and the allele for blood group N shows
blood group MN
(e) A person with the allele for brown eyes and the allele for blue eyes has brown eyes
15. If an alelel R is dominant over r

(a) Determine how many different phenotypes are present in the progeny of a cross
between Rr and rr, and in what ratio they are formed.
(b) Determine how many phenotypes are there, and in what ratio are they formed, if
there is no dominance between R and r.
16. A woman of blood group AB marries a man with blood group A. There are three
children in the family with blood groups A, B and O. (The allele for group A is codominant
to that for group B while the allele for group O is recessive to each of groups A and B
alleles). Identify which one of the children is adopted.

17. Work out the gametes that can be formed by an organism of each of the genotypes below:
(a) Aa (b) Bb (c) AaBb
18. In peas, tall plant habit is dominant over dwarf. If a plant homozygous for tall is
crossed with one homozygous for dwarf, state be the appearance of the (a) F1 (b) F2 (c) cross
of F1 with its tall parent plant and (d) with its dwarf parent
19. Short hair in rabbits is governed by a dominant gene (L) and long hair by its recessive
(l). Black hair results from the action of its dominant gene (B) and brown from the recessive
genotype (bb).
(a) In crosses between dihybrid short, black and homozygous short, brown rabbits,
work out the expected genotypic and phenotypic ratios expected among the
progeny.
(b) Determine the expected genotypic and phenotypic ratios in progeny from the cross
LlBb x Llbb using the fork-line method.
20. The most common form of human albinism is determined by homozygosity for the recessive
allele c. Persons homozygous for this recessive allele do not produce the enzyme tyrosinase,
which catalyses the first steps in the synthesis of the pigment melanin from the precursor
tyrosine. A normally pigment couple, each of whom has an albino parent are married and
have two children.
(a) Work out the probability that at least one of the children will be albino
(b) Work out the probability that both will be albino
21. Coat colours of the shorthorn breed of cattle represent a classical example of codominant
alleles. Red is governed by the genotype CRCR, white by CWCW and roan (a mixture of red
and white) by CWCR.
Predict the genotypic ratios expected among the progeny of a cross of roan shorthorns
among themselves.
22. Develop a general formula which expresses the number of different types of gametes
which can be formed in an organism with k pairs of chromosomes?
23. Using illustrations, explain a monohybrid test cross and a dihybrid test cross.
24. Define autosomal linkage and explain how it affects the phenotypic ratios in the dihybrid
cross.
25. Explain how you can use the chi-squared test to determine whether two genes are linked or
not.

8.10 Summary

Classical Mendelian genetics is based on the Laws of Heredity which were proposed by Gregor
Mendel. It explains the genetic basis of cell growth and cell division and lays the foundation for
statistical analysis of genetic data. The fundamental genetic terms are also discussed.
TOPIC 9 POST-MENDELIAN GENETICS

9.1 Introduction

After Mendel’s Law of Heredity were accepted by the scientific community, further research
revealed new phenomena which Mendel did not experience in his work. These post-Mendelian
phenomena include: gene linkage and recombination, multiple allelic genes, polygenes, lethal
genes and gene interactions. The study of these conditions constitutes Post-Mendelian Genetics.

9.2 Objectives: At the end of this topic you should be able to:
1. Identify the circumstances when Mendel’s first and second laws do not apply.
2. Explain gene linkage and recombination and their effects of on Mendelian phenotypic ratios.
3. Define multiple alleles and explain their effect on Mendelian phenotypic ratios.
4. Define lethal genes and explain their effects on Mendelian phenotypic ratios.
5. Define polygenes and explain their effect on Mendelian genetics.

9.3 Autosomal linkage and gene recombination

Genes which are located on the same chromosome are said to be linked and they show linked
inheritance. Such cannot behave independently of one another in their inheritance. Alleles that
are far apart will have higher chances of crossing over than those which are close together. This
is because for the alleles to crossover, there is need for the formation of a chiasma between them.
Unlinked genes located on different chromosomes assort quite freely and segregate their alleles
into gametes in all possible random combinations. This is a result of segregation and independent
assortment. Linked genes assort much less freely. The linking together of certain genes on the
same chromosome gives some control over recombination meaning that certain combinations of
characters can be kept together. This is a result of crossing over in which a chiasma is formed
between homologous chromosomes during meiosis.

9.4 Genetic recombination and variation

The F1 offspring from two individuals with different (pure) genotypes but of the same species
show genotypic and phenotypic variability which results from the reshuffling of alleles in the
gametes of their parents. The variation that comes about by recombination of alleles is the main
function of meiosis and sexual reproduction. This variation is important to the long term survival
and evolution of the species.
The F2 individuals with new combinations (which are absent in the parental types) are called
recombinants and are a result of crossing over and due to independent assortment during meiosis
in the F1 gamete formation. In general, the following genotypes and phenotypes are observed:
(i) The F1 of a monohybrid cross (n =1) will comprise three genotypes (3n) and two phenotypes
(2n). With independent segregation the phenotypes will be in the ratio 3:1. (ii) The F2 of a
dihybrid cross (n =2) will comprise nine genotypes (3n ) and four phenotypes (2n). With
independent segregation the phenotypes will be in the ratio 9:3:3:1.

Question: Predict the number of genotypes and phenotypes of the F2 of a trihybrid cross.
Solution: The F2 of a trihybrid cross (n =3) will have 27 (3n) genotypes and eight (2n)
phenotypes.

9.5 Lethal alleles

Lethal genes are essential genes which have alleles that cause an organism to die only when
present in the homozygous condition. There are a number of classes of lethal genes. (i) The
completely fully dominant lethal allele kills the carrier in both homozygous and heterozygous
conditions at the zygote stage, embryonic stage or even after birth or hatching. No individual
attains the age of reproduction. (ii) The recessive lethal allele will cause death in the homozygote
for the allele. Most lethal genes are recessive (iii) Sub lethal or semi lethal genes become
operative at the time the individuals become sexually mature. Such lethal genes cause some
handicap but do not destroy the affected organism.

9.5.1 Effects of lethal genes on Mendelian ratios

Lethal genes will eliminate one or more phenotypic classes from the adult progeny, thus
modifying the phenotypic ratio. For example, the 3:1 phenotypic ratio resulting from a
monohybrid cross will become 2:1 if the recessive allele is lethal.

i) Yellow fur allele (Y) in mice is a dominant lethal allele which causes death in homozygotes. It
has been observed that when two yellow mice are crossed, the offspring have a phenotypic ratio
of 2 yellow : 1 grey instead of the Mendelian 3:1 ratio.
F2 ¼ YY (aborted) ½ Yy (yellow) ¼ yy (grey)

(ii) Achondroplastic dwarfism allele (A) in man is a dominant lethal allele.

F2 ¼ AA (aborted) ½ Aa (dwarf) ¼ aa (normal)
The phenotypic ratio observed in the F2 is 2 dwarf: 1 normal (2:1)

(iii) Chlorosis allele (C) in maize which results in plants lacking chlorophyll (chlorotic
plants).
F2 ¼ CC (die early) ½ Cc (chlorotic plants ) ½ cc (normal plants)
The phenotypic ratio observed in the F2 is 2 chlorotic: 1 normal (2:1)

9.6 Multiple alleles

In Mendelian inheritance, genes have only two alleles, such as a and A. In nature, a number of
genes exist in more than two allelic forms and are therefore said to have multiple alleles. Any
given individual will only have two copies of each gene, but the different alleles are found within
a population and hence the F2 generation will have more than three phenotypes. Many genes
have multiple alleles, including the human genes for ABO blood type.

9.6.1 The ABO blood group system

The ABO blood group system is one of several different blood group systems in man and is a
good example of multiple allelic inheritance. Blood group phenotypes are due to proteins, bound
to the surface of the red blood cells which act as antigens. In the ABO system the blood groups
are controlled by a single gene, designated as I, which has three alleles IA, IB and IO. IA is co-
dominant to IB ; IA dominant to IO; IB dominant to IO (IA = IB > IO). There are six possible pair-
combinations of the alleles, giving six genotypes as shown in the table below:

Table 9.1 ABO blood group phenotypes and genotypes. Source:

Blood group Genotypes

phenotypes
A IAIA
IAIO
IBIB

B IBIO
AB IAIB
O IOIO
9.6.1.1 Inheritance of blood groups

Blood groups are inherited in the Mendelian fashion but each individual will only carry two out
of three alleles.

Examples of crosses involving the ABO blood groups are given below.

(i) Parents ♀Group O x ♂Group O

Genotypes ♀ IOIO x ♂IOIO

All children will be blood group O

(ii) Parents ♀Group AB x ♂Group O

Genotypes ♀ IAIB x IOIO ♂

Gametes ½ IA, ½ IB All IO

Children ½ IAIO ½ IBIO

½ Group A ½ Group B

Genotypic ratio: ½ IAIO: ½IBIO

Phenotypic ratio: ½ A: ½ B

(iii) Parents ♀ Group AB x Group AB ♂

Genotypes ♀ IAIB x IAIB ♂

Gametes ½ IA, ½ IB ½ IA, ½ IB

The possible offpspring are shown in the Punnet square below.

♀/♂ ½ IA ½ IB
½ IA ¼ IA IA ¼ IAIB
½ IB ¼ IAIB ¼ IBIB

Genotypic ratio: ¼ IA IA: ½ IAIB: ¼ IBIB

Phenotypic ratio : ¼ A: ½ AB: ¼ B
9.6.1.2 Red blood cell antigens and serum antibodies

The surfaces of red blood cells contain antigens while the blood serum contains natural
antibodies against foreign RBC antigens. The antigen-antibody combinations for all the possible
blood groups are shown in Table 9.2.

Table 9.2 ABO blood groups and their associated antigens and antibodies.

Blood group (phenotypes) Genotypes RBC antigens Serum antibodies

IAIA A

A IAIO A Anti-B

IBIB B

B IBIO B Anti-A

AB IAIB AB No anti-A, no Anti-B

O IOIO No antigens Anti-A and anti-B

When a blood group carrying a given antigen is mixed with a blood group carrying an antibody
for that particular antigen, the blood cells in the mixture agglutinate (clump together).

9.6.1.3 Blood typing

The results of the reactions used for blood typing are summarised in Table 9.3.

Table 9.3 Blood typing reactions.

Reaction to serum according to blood group

Serum type O A B AB

Anti-A serum - + - +
Anti-B serum - - + +

A positive result represents agglutination; a negative result represents no agglutination

9.5.4 Blood transfusion

When small quantities of blood are transfused some groups can be safely mixed with others as
shown in Table 9.4.

Table 9.4 Safe and unsafe blood transfusion.

Recipient

O A B AB
Donor Anti-A + Anti-B Anti-B Anti-A No antibody
antibodies antibody antibody

O (No antigen) - - - -

A (A antigen) + - + -

B (B antigen) + + - -

AB(A+B antigens) + + + -

The + sign represents agglutination meaning transfusion is not safe; the – sign represents no
agglutination meaning transfusion is safe.

Group O can donate to any of the other groups because O has no A or B antigens on its red blood
cells and cannot be agglutinated by antibodies in the recipient’s serum. For this reason people of
group O are referred to as universal donors.

AB blood has no anti-A or anti-B antibodies and can receive blood from any of the other groups
because it cannot agglutinate their red cells. Persons of group AB are therefore known as
universal recipients.

9.6 Polygenic inheritance

A study of the phenotypic differences in any large population shows that two forms of variation
occur namely (i) discontinuous variation and (ii) continuous variation.
9.6.1 Discontinuous variation

In this kind of variation individuals show clear-cut differences with no intermediates between
them. Examples of discontinuous variation include blood groups and albinism in humans, wing
lengths in Drosophila, melanic (dark) and light body colour in the moth Biston betularia, and sex
in animals and plants. Characteristics showing discontinuous variation are usually controlled by
one or two major genes which may have two or more allelic forms and their phenotypic
expression is relatively unaffected by environmental conditions. This form of variation is also
known as qualitative variation.

9.6.2 Continuous variation -Polygenic inheritance

This is a type of inheritance in which one character is controlled by many genes (polygenes)
working together to bring about the phenotype. Each gene has two or more alleles. It involves
characters that show a range measuring from one extreme to the other within a population.
Characters exhibiting continuous variation are controlled by the combined effects of many genes
and the environment. The frequency distribution for a character showing continuous variation is
a normal curve. Most of the organisms in the population fall in the middle of the range with
approximately equal numbers showing the two extreme forms of the characteristic. Individually
each of these genes has little effect on the phenotype but their combined effect is significant.
Polygenic characters are also called quantitative traits or continuously varying traits.
Although polygenic traits are inherited in the Mendelian fashion, they are studied by use of
statistical methods dealing with populations.

In humans, height is controlled by polygenic inheritance. An individual may possess the

genotype for being very tall height, but if he or she does not eat nutritious foods during the early
years of growth, he/she will not be able to grow as tall as the genotype would permit.
In plants, yield is a polygenic trait which is affected by many genes controlling factors such as
rate of germination, rate of photosynthesis, amount of root, drought tolerance, etc.
Figure 9.1. Normal distribution of a polygenic trait

The differences between qualitative and quantitative are summarised in Table 9.5.
Table 9.5. Comparison between qualitative and quantitative traits
Qualitative traits Quantitative traits
1 Characters are of a kind Characters are of a degree
2 Show discontinuous variation Show continuous variation
3 Characters controlled by single genes in which Polygenic control in which each gene has a
each gene has a major effect on the character small effect
4 Changing one gene for another at a locus Changing one gene for another one at a locus
causes a major difference in the phenotype causes relatively little difference in the
phenotype
5 Based on individual mating events between Based on populations in which mating events
couples and their progeny take place at random and many different sets of
offspring are produced
5 Analysed by making counts and simple ratios Analysed by use of statistics using the chi-
squared test and other tests
6 The environment has a small effect on them The environment has a major effect on them
7 Examples; i) sweat pea flowers are either Examples (i) height, intelligence, skin colour,
white or pink, (ii) the sex of a person is either beauty are examples of human quantitative traits
male or female, (iii) the blood group of a (ii) crop yield (productivity) is a plant
person is A or B or O or AB. quantitative trait

9.7 Gene interactions

In many cases genes at different loci interact with each other leading to unexpected phenotypes.
These non-allelic gene interactions include epistasis, hypostasis, complementary action and
pleiotropy.

9.7.1 Epistasis

Epistasis means ‘standing upon’. This is the interactions between two different genes where one
gene hides the expression of another gene. The epistatic gene interferes with the phenotypic
expression of another (hypostatic) gene. Epistasis can be dominant or recessive.
9.7.1.1 Recessive epistasis

One form of coat colour in mice provides an example of recessive epitasis. The hair colour of the
wildtype mice is described as agouti in which there is a yellow band around an otherwise black
hair. The agouti allele (A) is dominant to black allele (a) and therefore individuals with the
genotype AA or Aa are agouti whilst individuals with the genotype aa have black hair. Another
independently inherited gene is required for the synthesis of the hair pigment. This gene has two
alleles i.e. dominant allele C which is responsible for colour expression and recessive allele c
which is responsible for non-expression of colour. Individuals of genotypes CC and Cc are
coloured while cc individuals are albino (no colour). It has been observed that the individual with
the genotype AAcc will be albino because cc is epistatic to the colour gene. The F2 generation of
mice with these two gene is give in the table below:
Table 9.6. Recessive epistasis in coat colour in mice (AaCc x AaCc). Source:
AC Ac aC Ac
AC AACC AACc AaCC AaCc
Agouti Agouti Agouti Agouti
Ac AACc AAcc AaCc Aacc
Agouti albino Agouti Albino
aC AaCC AaCc aaCC aaCc
Agouti Agouti Black Black
Ac AaCc Aacc aaCc aacc
Agouti albino Black albino
Phenotypic ratio = 9 agouti: 3 black: 4 albino (Modified 9:3:3:1 ratio)
9.7.1.2 Dominant epistasis

Dominant epistasis is seen in the inheritance of plumage (feather) colour in chickens. Pure-
breeding White Leghorns are homozygous dominant for the gene I which causes inhibition of
colour in the feathers. They are also homozygous for the gene C which brings about coloured
feathers. The IICC chickens are white because allele I is epistatic over C. Pure-breeding White
Wyandotte chickens are homozygous for carry the recessive i for colour inhibition and also
homozygous for recessive allele c for lack of colour in feathers. The iicc chicken are white
because genotype ii inhibits colour in feathers and the genotype cc is responsible for lack of
colour . When White Leghorns are crossed with White Wyandottes (I ICC x iicc) the F1 are all
white (IiCc). Interbreeding among the F1 produces F2 which consists of white and coloured
chickens in the ratio 13: 3. The genotypes containing II or Ii are white and the double recessive
iicc is also white. Only the iiCC and iiCc are coloured. Here the colour inhibiting gene I is
epistatic to the colour producing gene C. Individuals that carry the dominant allele I will have
white feathers even if they are carrying the dominant allele C for colour production. For example
an individual with the genotype IiCC will be white and individuals which are homozygous
recessive for the colour gene (cc) will also be white.

Table 9.7. Dominant epistasis in plumage colour in chickens (IiCc x IiCc). Source:

IC Ic iC Ic
IC IICC IICc IiCC IiCc
white white White White
Ic IICc IIcc IiCc Iicc
white white White White
iC IiCC IiCc iiCC iiCc
white white coloured coloured
Ic IiCc Iicc iiCc iicc
white white coloured white
Phenotypic ratio = 13 white : 3 coloured (modified from the 9:3:3:1 ratio)

9.7.2 Hypostasis

Hypostasis is a situation in which a (hypostatic) gene is hidden by another (epistatic) gene.

9.7.3 Pleiotropy
Also known as non-epistatic interaction, pleiotropy explains the multiple phenotypic effects of a
single gene. Genes that affect a fundamental process will affect many other processes in a
complex organism.
There are two ways in which a single gene may affect several different characters. The first is
that the gene produces a product that involved in a branched biochemical pathway, and a
mutation in the gene therefore affects different branches. The second cause of pleiotropy is when
the gene determines an enzyme, which is common to a number of different metabolic pathways.
Mutation in the gene will then cause a block in these otherwise unrelated pathways.

For example sickle cell anaemia is due to a mutation in one gene which leads to the production
of abnormal haemoglobin. The effects of the abnormal haemoglobin are many and complex and
include the sickle-shape of the red blood cells and their tendency to clump together and clog
blood vessels in various organs of the body. As a result, heart, kidney, spleen and brain damage
are common elements to the syndrome. The defective red blood cells are also readily destroyed
in the body, causing severe anaemia. Another example of Pleiotropy is albinism.

9.7.4 Complementary genes

Complementary genes interact together to produce an effect which is different from that given by
either of them separately. One unusual gene interaction was observed by Bateson and Punnett in
the character comb type in chickens. There are a number of different forms of the comb, two of
which are called ‘pea’ and ‘rose’. Both of these types breed true, but when crossed together the
resultant F1 hybrid has an entirely different form called ‘walnut’. When walnut birds interbreed,
the F2 generation has four comb types: walnut, pea, rose and a new type known as single. The
four phenotypes occur in the ratio 9:3:3:1 (Figure 10.2 and Table 10.2).
Figure 10.2 Pea comb hen crossed with a rose combed cock: For the F2 generation (See
Table 9.2)

Table 9.2 The progeny of the F2 generation.

1/ PR 1/ Pr 1/ pR 1/ pr
4 4 4 4

1/ PR PPRR PPRr PpRR PpRr

4

walnut walnut walnut walnut

1/ Pr PPRr PPrr PpRr Pprr
4

walnut pea Walnut pea

1/ Pr PpRR PpRr ppRR ppRr
4

walnut walnut rose rose

1/ pr PpRr Pprr ppRr Pprr
4

walnut pea rose single

Phenotypic ratio is 9 walnut: 3 pea: 3 rose: 1 single
Bateson and Punnett explained these results on the basis of the interaction of two independent
genes which were called P (for ‘pea’) and R (for ‘rose’). Pea and rose are both dominant over
single. A genotype which has at least one dominant allele of the gene P has a pea comb, and one
with at least one dominant allele of the gene R has a rose comb. A genotype without any
dominant alleles has a single comb, which results from the interaction between two homozygous
recessive pairs of alleles. When the dominant alleles of both P and R are present together, they
interact to give the walnut phenotype. Pure breeding parents with pea combs are therefore of
genotype PPrr and those with rose combs are ppRR. The F1 are walnut because they have the
dominant alleles of both genes (PpRr). The important clue in this cross is the way in which the
different phenotypic classes are seen to be in multiples of 1/16ths. This confirms that the F2 must
have come about by the crossing of F1s which were heterozygous for two pairs of independently-
segregating genes.

Although the phenotypic ratio is 9:3:3:1, it differs from the normal dihybrid cross because in this
case there is only one character involved which is comb form.

9.8 Revision exercise

1. Define lethal alleles.

2. State the modified Mendelian ratios caused by epistasis and lethal alleles.
3. Explain how multiple allelic characters affect the Mendelian genetics.
4. Distinguish between single genes and polygenes.
5. Explain how gene linkage affects the Mendelian phenotypic ratios.
6. In the fruit fly, vestigial (rudimentary) wings are determined by a recessive allele v. In
one experiment a number of crosses Vv x Vv yielded the following phenotype in the
indicated numbers:
Normal wings 378
Vestigial wings 121
(a) State which simple ratio this outcome represents.
(b) State whether this ratio deviates from the expected Mendelian ratio
(c) If there is a deviation, state the possible cause.
7. Chickens with shortened wings and legs are called creepers. Crosses between normal
chickens produce only normal progeny. When creepers were mated to creepers their
offspring comprised 23 creepers and 11 normal chickens.
(a) State which simple ratio this outcome represents
(b) State whether this ratio deviates from the expected Mendelian ration
(c) If there is a deviation, state the possible cause.
8. Dominant lethal alleles are very rare and when they arise, individuals carrying them
normally disappear rapidly from the population. Explain why.
9. Compare and contrast between dominance and epistasis.
10. In humans, a certain allele results in severe anaemia plus damage to the kidneys and
central nervous system. State the genetic phenomenon illustrated by the expression of this
allele.
11. Hull colour in oat seeds is determined by two genes. When a plant with the genotype
AaBb is crossed with another of the same genotype, their offspring presents the following
phenotypes in the indicated numbers:
Black hulls 430
Gray hulls 106
White hulls 36
(a) State which simple ratio this outcome represents
(b) State whether this deviates from the expected Mendelian ratio
(c) If there is a deviation, is it caused by lethal alleles, epistasis, variable expressivity or
incomplete penetrance? Justify your answer.
12. In poultry, the shape of the comb varies greatly and involves at least two pair of
alleles. The allele R can result in rose comb, and the P can result in pea-shaped comb. If both
of these dominants are present, genic interaction produces a walnut comb. When a bird is
carrying both recessives r and p, in the homozygous condition, a single comb type results.
(a) Give the phenotypes of each of the following pairs of birds and the phenotypic ratio
to be expected among their offspring as to comb type.
(i) RrPp x RrPp
(ii) RRPp x rrPp
(iii) rrPP x RrPp
(b) In the following question determine the genotypes of the parents. A rose crossed
with a walnut produces offspring three-eighths of which are walnut, three eighths
rose, one eighth single.
13. The coat colours of Labrador retriever dogs are black (determines by the genotype
B_E_), brown (bbE_), or yellow (_ _ ee). If dihybrid black dogs are mated (BbEe x
BbEe), determine the phenotypes expected in the progeny and their proportions.

9.9 Summary
It has been observed that in some crosses, it is not possible to get the phenotypic ratios predicted
by Mendelian Genetics. The results deviate from the expected Mendelian ratios. Careful analysis
of these deviations has shown that they are caused by various reasons which include: gene
linkage and recombination, multiple allelic genes, polygenes, lethal genes and gene interactions.
The study of these conditions constitutes Post-Mendelian Genetics.
10. SEX DETERMINATION AND SEX LINKAGE

10.1 Introduction

Sexual differentiation of a species into separate male and female forms is usually genetically
determined. The inheritance of sex can be interpreted in Mendelian terms as a monohybrid test
cross. Mating between males and females always results in two sexes among the offspring in
approximately equal numbers. Sex chromosomes are those that carry the sex-determining genes.
All the other chromosomes which are not involved in sex determination are known as autosomal
chromosomes or autosomes.

In Humans and many other animals sex is determined by the presence of different sets of sex
chromosomes. Women have two X-chromosomes while men have one and one Y- chromosome.
In most animal species, the Y-chromosome only contains the genes responsible for the
development of the male organs producing the male gametes, the testes. The Y-chromosome is
therefore considered to be genetically empty. In these cases, the Y- chromosome is also
physically much smaller than the X-chromosome. The above situation means that gene located
on the X-chromosome (also called sex-linked genes) are present with only one allele in the male
individual, because the male has only one X-chromosome. In females the sex-linked genes are
present with two alleles (one allele on each X-chromosome), like happens with all genes located
in autosomal chromosomes. As a consequence, recessive, sex-linked diseases caused by
recessive alleles occur more frequently in males than females.

10.2 Objectives
1. State the chromosomes involved in sex determination.
2. Differentiate between the X and Y chromosomes.
3. Define sex-linked, sex-influenced and sex limited genes
4. Explain the hormonal effects on sex expression.
5. Explain sex-linked inheritance of feather colour in birds.
6. Discuss sex-linked diseases in humans.
10.3 Inheritance of sex in animals
In human and many other animals, sex is determined by the presence of different sets of sex
chromosomes designated X and Y. Females have two X chromosomes; males have an X and a Y
chromosome. In most species, the Y chromosome only contains the genes responsible for the
development of the male organs producing the male gametes, the testes. It is therefore considered
to be genetically empty. In most cases the Y chromosome is physically smaller than the X
chromosome. The female is therefore homogametic (XX) producing eggs carrying a single X
while the male is heterogametic (XY) with half the gametes carrying the X chromosome and the
other half carrying the Y chromosome.

In the XX/XY system therefore, the sex of the offspring is determined by the male parent.

Figure 10.1 Inheritance of sex in animals

10.4 Sex (X-chromosome) linked inheritance

Sex chromosomes also carry genes which are not directly involved with the determination of sex.
These so-called sex-linked genes are carried on the X chromosome and are present with only one
allele in the male sex. In females the sex-linked genes are present with two alleles (one on each
X-chromosome) like what happens with all genes located on autosomes. As a result, recessive,
sex-linked alleles will always be expressed in the male’s phenotype, because he will never have
the dominant counterpart to mask its effect. That is the reason why sex-linked diseases caused by
a recessive allele occur more frequently in men than in women.

In the XX/XY system, there are genes in the differential part of the X chromosome which have
no allelic partners in the Y and vice versa. These are the sex-linked genes.

Figure 10.2 The X and Y chromosomes. Source: Jones and Karp. 1994. Introducing Genetics.
Page 98.

Examples of sex-linked genes include those for (i) haemophilia in humans and (ii) colour
blindness in humans

Two indications of sex-linkage:

(1) When a recessive allele appears more frequently in males than in females in a pedigree.

(2) When a reciprocal cross yield a different phenotypic ratio.

10.5 Haemophilia in humans

Haemophilia is a human disease in which the main feature is a reduced ability of the blood to
clot. Haemophiliacs can therefore bleed excessively even from very small wounds. The
commonest form of haemophilia is caused by a recessive allele h of a single gene ‘H’ located on
the X chromosome. ‘Normal’ parents can have haemophiliac children as illustrate below:
Figure 10.3 The offspring of Queen Victoria and their risk of haemophilia. Source: Jones
and Karp. 1994. Introducing Genetics. Page 101.

Although Queen Victoria was phenotypically normal, she carried the allele for haemophilia
which she passes on to her children of which only her sons could be haemophiliac.

10.6 Colour blindness in humans

Colour blindness is the inability to distinguish between green and red. This condition results
from a recessive allele c of gene C carried on the X chromosome. This condition is more
frequent in males than females for obvious reasons.
The inheritance of colour blindness can be illustrated as given below:

Figure 10.4 Inheritance of colour blindness. Source: Jones and Karp. 1994. Introducing
Genetics. Page 103.

10.7 Feather colour in birds

In birds, the male is heterogametic while the female is homogametic. The same principle of
inheritance applies as the one for sex linkage in humans but the pattern of inheritance is reversed
in the sexes.

In one example, Light Sussex chickens have mostly white plumage and the Rhode Island Red
breed have mostly red. The allele R for white feathers is dominant to the allele r for red feathers.
A cross between Rhode Island cockerels and Light Sussex hens produces white male offspring
and red female offspring as shown below:
Figure 10.5 Inheritance of feather colour in birds. Source: Jones and Karp. 1994. Introducing
Genetics. Page 105.

10.8 Holandric genes or Y-linked genes

These are genes located on the Y-chromosome whose effects only pass to male offspring from
their father e.g. the pattern baldness allele and the hairy ear (hypertrichosis) allele.

10. 9 Hormonal effects on sex expression

The gender of an animal can also influence or limit the effects or expression of certain sex-
influenced genes. These genes are not necessarily located on the X chromosome, most of the
times they are even on the autosomes. The cause of these gender-mediated differences in gene
expression is the difference in level of male and female sex hormones between genders.
10.10 Sex-influenced (sex-affiliated) genes

When the same genotype leads to different phenotypes in male and female, the gene involved is
said to be sex-influenced (sex-affiliated). For example, the allele responsible for pattern
baldness (androgenetic alopecia) in humans is expressed more frequently in males than in
females. The allele responsible is dominant in males and recessive in females.

Table 10.1. Inheritance of pattern baldness. Source:

Genotype Male Female

BB Bald Non-bald
Bb Bald Non-bald
Bb Non-bald Bald

The allele B in both the homozygous and heterozygous states exerts its dominance only in the
male sex while in the female only the homozygous BB exerts dominance.

10.11 Sex-limited genes

Usually sex-limited characters are expressed only in one sex even though both sexes may carry
genes for such characters. The expression of these genes is determined by the presence or
absence of sex hormones e.g. beards in man and milk production in females. Both men and
women possess the genes for producing milk, but only in men these genes are never expressed.
Sex-limited genes may be carried on autosomes and are strongly affected by the level and types
of sex hormones present in the body.

10.12 Revision exercise

1. The gene for eye-colour in the fruit fly, Drosophila comes in many different alleles
(e.g. white, orange, ruby, pink, and red). The gene is present on the X-chromosome but not on
the Y-chromosome. Sex determination in Drosophila is the same as in man.
(a) Work out how many allele of this gene one male individual
(b) Work out how many allele of this gene one female individual
2. State whether or not a human male possess genes for producing baby milk
3. A narrow reduced eye called “bar” is a dominant sex-linked condition in Drosophila
due to the allele B. Normal eyes are produced by the recessive allele b.
(a) A homozygous female with normal eyes is mated with a bar-eyed male. Predict the
genotypic and phenotypic ratios of this cross, including the sex.
(b) Predict the genotypic and phenotypic ratios of the reciprocal cross, including the sex.
4. A sex-linked recessive allele c produces a red-green colour blindness in humans. A normal
woman whose father was colour blind marries a colour blind man.
(a) Predict the genotypes that are possible for the mother of the colour blind man
(b) Work out the chances that the first child from this marriage will be a colour blind boy
(c) Of the girls produced by this couple, predict the proportion that can be expected to
have normal vision.
5. The sex-influenced allele governing the presence of horns in sheep is dominant in males, but
recessive in females. Its counterpart, the allele for being hornless is recessive in males and
dominant in females. Let “H” represent the allele for having horns and let “N” represent the
allele for being hornless. A monohybrid cross is executed.
(a) State the phenotype of the male and of the female
(b) Predict the genotypic and phenotypic ratios for a monohybrid cross.
6. In humans, the autosomally linked allele A is required for normal skin pigmentation.
Its recessive allele a is associated with the albino condition. Several pairs of alleles
affect colour vision. One of the X-linked recessives responsible for red-green colour
blindness may be represented as p. Its dominant allele P is required for normal colour
vision. Write as much as possible of the genotypes of the following three persons:
(a) A woman with normal vision and normal skin pigmentation whose father is a colour-
blind albino.
(b) A normally pigmented man whose mother is colour blind and whose father is albino.
(c) A man with normal vision and normal skin pigmentation whose father is a colour-
blind albino.

10.13 Summary

In animals, the sex of the individual is determined by the X and Y chromosomes. XX individuals
are homogametic and are usually female while XY individuals are hetrogametic and are usually
male. In addition to the chromosomes, hormones also have an influence on the expression of
certain sexal characteristics. There are a number of human genetic diseases which are linked to
the sex of the affected person.
TOPIC 11 CHROMOSOME MUTATIONS

11.1 Introduction

Chromosome mutation is the processes of change in chromosomes Chromosome mutations are

of two types i.e. (1) changes in chromosome structure and (2) changes in chromosome numbers.
Structural mutation may be due to deletion (deficiency), duplication, translocation or inversion.
The major two types of mutation in chromosome number are euploidy and aneuploidy.
Chromosomal mutation lead to phenotypic abnormalities in the affected individuals are
responsible for a number of human genetic diseases.

11.2 Objectives: At the end of this topic you should be able to:

1. Explain mutations in chromosomal structure.

2. Explain mutations in chromosomal structure.
3. Discuss human diseases caused by chromosomal mutations.
4. Describe methods of detection of human genetic diseases.
5. Describe methods of pedigree analysis of human genetic diseases.

11.3 Mutations in chromosome structure

Structural mutation may be due to deletion (deficiency), duplication, translocation or inversion. These
changes in chromosomes lead to formation of loops during meiosis. The chromosome set mutates
spontaneously four types of rearrangements namely: deletions, duplications, inversions and
translocations.

11.3.1 Deletion (deficiency)

Represent a loss in chromosomal material, which may carry one or more genes. Consider
chromosome abcdefg below which loses a part fg through deletion.
Figure 11.1 . Deletion of fg from chromosome abcdefg.

A deletion heterozygote can lead to pseudo-dominance in which an allele is deleted from one
chromosome making the alternative recessive allele on the intact chromosome express itself as if
it were dominant.

In humans, a deletion of a large part of the short arm of chromosome 5 causes a genetic disease
called ‘Cri-du Chat syndrome’. The affected babies have a cat-like cry, are also mentally
retarded, have a moon face, low birth weight, saddle nose, small mandible and malformed low-
set ears. Partial deletions of chromosomes 4, 13 and 18 are also known to cause specific
syndromes.

12.3.2 Duplication (addition)

The presence of a section of a chromosome in excess of the normal amount is known as

duplication. The repeated section of chromosomal material may be present in one pair of
homologous chromosomes or may have been transposed to a nonhomologue. Generally
speaking, duplications are not as deleterious as deletions. In fact duplications usually act as new
sources of genetic variation. The diagram below illustrates duplication of segment ab.

Figure 11.2. Duplication of ab in chromosome abcdefg

11.3.3 Translocation

A translocation moves a chromosome segment to another position in the genome. Chromosomes

sometimes undergo spontaneous rupture, or can be induced to rupture due to high frequency of
ionisation radiation. The broken ends of such chromosomes may re-join into a non-homologous
position. New gene linkages can be produced by translocations. Translocation heterozygotes may
have 50% reduced fertility. Translocations are an important cause of ill health in human
populations.

11.3.3.1 Simple translocation

Involves a single break in the chromosome and the transfer of a broken piece directly onto the
end of another.

Figure 11.3. Simple translocation of bd from abcdefg to one end of vwxy

11.3.3.2 Shift (intercalary) translocation

Involves three breaks, so that a middle piece from one chromosome is inserted within the break
in a nonhomologous chromosome.
Figure 11.4. Shift translocation of bc in between x and y involving nonhomologues.

11.3.3.3 Reciprocal translocation (interchanges)

These occur when single breaks in two nonhomologous chromosomes produce an exchange of
chromosome segments.

Figure 11.5. Reciprocal translocation of segments bc and wx between two homologues

The Philadelphia chromosome is caused by the reciprocal translocation between chromosome

9 and 22. It is found in about 90% of patients with chronic myelocytic leukaemia (a blood
cancer). t(9;22)(q34;q11) - Philadelphia chromosome, Chronic myeloid leukaemia (CML), ALL
Reciprocal translocation between chromosomes 8 and 14 causes most cases of Burkitt’s
lymphoma, a cancer of the B lymphocytes. t(8;14) - Burkitt's lymphoma (c-myc)

11.3.4 Inversion
An inversion involves a break of segment in a chromosome and the rejoining of the segment but
after a 180° turn in the segment.
.
Figure 11.6. Inversion of the segment cd.

Pericentric inversions span the centromere while paracentric inversions do not span the
centromeres.

Figure 11.7. Paracentric and pericentric inversions.

An inversion can disrupt a gene if it occurs in the middle of a gene and generally speaking
paracentric inversions are less deleterious than pericentric inversions.

11.4 Mutations in chromosome numbers

Each species has a characteristic number of chromosomes. Humans and higher organisms are
diploid (2n), with two sets of homologous chromosomes: one set donated by the father, the other
set by the mother. Mutations in the number of sets of chromosomes (ploidy) are also commonly
encountered in nature. About one-third of the flowering plants (angiosperms), for example, have
more than two sets of chromosomes. There are two types of variation in chromosome number
namely (1) Euploidy and (2) Aneuploidy
11.4.1 Aneuploidy

Aneuploidy is the change in number of individual chromosome. Chromosomes may be added to

or subtracted from normal sets. One or more whole chromosomes may be absent from or may be
added to the chromosome set (complement). The cause of aneuploidy may be due to a process
called non-disjunction (Figure 11.8). Non-disjunction is a mistake in which both chromosomes
(or chromatids) pass to the same pole of a cell instead of opposite poles at anaphase during
meiosis.

Figure 11.8. The origin of aneuploid gametes by nondisjunction at the first or second
meiotic division. Source: Griffiths et al. 1993. Introduction to Genetic Analysis. Page 251.

Aneuploid conditions are well studied in humans. Down syndrome (Trisomy 21), Klinefelter
syndrome (XXY), and Turner syndrome (XO) are well documented. The spontaneous level of
aneuploidy in humans is quite high and produces a major proportion of genetic diseases in
human populations.
11.4.1.1 Non-disjunction of sex chromosomes

Failure of sex chromosomes in either sex to separate during meiosis in either male or female
individuals results in the production of both normal and rare aneuploid gametes (Figure 11.9).

Non-disjunction in the male Non-disjunction in the female

P: 46, AA XY ♂ P: 46, AA XX ♀

Gametes AX, AY, A0, AXY, AXX, AYY AX, AXX, A0

Normal With nondisjunction Normal With nondisjunction
Figure 11.9. The origin of aneuploid gametes in males and females

Fertilization of the rare gametes results in various sex chromosomal anomalies and associated
abnormal phenotypes in the offspring as shown in Table 12.1.

11.4.1.1. Trisomics (2n +1) - polysomy

There are several examples of viable trisomics in humans such as the Klinefelter syndrome
(XXY) in which the affected persons are sterile males with effeminate tendencies; and the XYY
syndrome in which the males affected are sterile and are physically normal but socially deviant.
The most common type of human aneuploidy is the Down syndrome, an autosomal trisomic
caused by non-disjunction of chromosome 21. There is a maternal-age effect for Down
syndrome; older mothers show a greater risk of having Down syndrome children. Facial features
include eyelids which apparently slant upwards due to a fold of skin over the inner corner of the
eye. The face is flat and rounded. Other symptoms include: mental retardation; short stature and
relatively small skull due to poor skeletal development; heart defects occur in about one-quarter
of Down’s children; increased risk of respiratory and ear infections; high risk of leukaemia;
coarse, straight hair.
Table 11.1. The F1 resulting from different types of nondisjunction of sex chromosomes
Genotype Characteristic
46, AAXX Normal female
Diploid
46, AAXY Normal male
Diploid
47, AAXXX Superfemale (metafemale) range phenotypically from normal females to nearly
Trisomic like those with Turner’s syndrome. Have a high incidence of mental retardation
47, AAXXY Klinefelter syndrome males: sterile, with long limbs, feminine breast
Trisomic development (gynecomastia) and mentally retarded
47, AAXYY Tall-aggressive syndrome: males. Fertile or infertile, usually very tall. Most of
Trisomic the very vicious murders in the world are committed by them
45, AAX0 Turner’s syndrome: infertile females with underdeveloped external genitalia. Has
Monosomic a webbing of neck skin, short stature, shield like chest and low intelligence
45, AAY0 Rare males, usually die.
Monosomic

12.4.2. Euploidy

Euploidy is variation in the number of whole sets (complements) of chromosomes. Diploids with
two sets of chromosomes in their nuclei are regarded as being the normal form in eukaryotes.

11.4.2.1 Polyploids

Organisms with three or more complement sets of chromosomes are known as polyploids. These
include triploids (2n=3x), tetraploids (2n=4x), pentaploids (2n=5x) and hexaploids (2n=6x).
Polyploids are common in plants e.g. bread wheat, Triticum aestivum, (2n=6x=42). Another
example of plant polyploidy is the potato, Solanum tuberosum (2n = 4x = 48). Polyploids are
rare in animals where they usually fail to develop and are aborted. Polyploids can arise due to
non-disjunction in which either: (i) two gametes with unreduced chromosomes fuse to form a
tetraploid zygote or (ii) a gamete with unreduced chromosomes fuses with a normal gamete to
form a triploid zygote.
The unreduced gametes are formed when there is failure to form spindles at anaphase during
meiosis resulting in diploid gametes.

11.4.2.1 Monoploids

These have only one set that develops from unfertilised eggs and are very rare. Male bees, wasps
and ants are monoploids which develop parthenogenetically from unfertilized eggs.

11.5. Human genetic diseases

Human genetic diseases are those that are inherited by individuals from their parents.

11.5.1 Detection of human genetic diseases

The first step towards the pedigree analysis of human diseases is the detection of the diseases
themselves. There are a number of techniques used to diagnose genetic diseases. Prenatal
diagnostic methods, for example, are used to detect disorders in unborn children. These
techniques include; amniocentesis, chorionic villi sampling and ultra sound.

11.5.1.1. Amniocentesis

In this method a sample of the amniotic fluid is taken using a syringe and a hypodermic needle
around the sixteenth week of pregnancy. It is separated into fluid and cells. The fluid is used to
detect (a) neural tube disorders and (b) biochemical (metabolic) diseases while the cells are
cultured and used to study (a) foetal sex, (b) chromosomal disorders and (c) metabolic diseases.
11.5.1.2. Chorionic villi sampling

In this technique a sample of finger like projections called chorionic villi is taken from the
chorion, which is the outermost membrane surrounding the foetus. The sample is taken using
either a transabdominal needle guided by an ultrasound probe or through a transcervical catheter
also guided by an ultrasound probe. This technique is used to study foetal sex, metabolic
disorders and chromosomal disorders. Although this method produces faster results compared to
amniocentesis, it cannot detect neural tube defects.

11.5.1.3. Ultra sound

Ultra sound is used together with amniocentesis and chorionic villi sampling in locating the
placenta, establishing an accurate gestational stage and excluding multiple pregnancy and foetal
death.

11.5.2 Pedigree analysis of human genetic diseases

A human pedigree is a chart of an individual's ancestors used in human genetics to analyze

Mendelian inheritance of certain traits, especially of inherited diseases.

The use of pedigree analysis involves collection of information about a particular family and
their ancestors. This information is then used to construct a pedigree chart (family tree). This
chart contains names of people and also information about their phenotypes. Using this chart it is
possible to predict the risks of a particular couple having a child who might suffer from a
particular genetic disorder.

The symbols used in pedigree analysis are summarised in Figure 6.1. Consanguinity is the
marriage of related individuals, for example cousins. The propositus is the individual who first
draws the geneticist’s attention to a particular family.
Figure 11.10. The symbols used in pedigree analysis. Source: Hayward.1992. Applied
Genetics. Page 198.

11.5.2.1 Autosomal dominant traits

Autosomal traits are carried on the autosomal chromosomes (not on the sex chromosomes).
Inheritance of autosomal traits is best-studied using dominant traits. Some clues used to show
that a trait is not sex-linked (X-linked) are:
(i) Equal numbers of male and female offspring are affected
(ii) If the affected male passes the trait to his sons, it means the gene cannot be on the X-
chromosome since the man only passes the Y chromosome to his sons.
(iii) If the affected male has affected daughters as well as unaffected daughters-because if
the gene were X-linked dominant then all his daughters would be affected, since he
transmits his X chromosome to each of them.

Figure 11.11. A pedigree showing a pattern of inheritance consistent with a trait inherited as an
autosomal dominant. Source: Hayward.1992. Applied Genetics. Page 199.
11.5.2.2 Autosomal recessive inheritance
An autosomal recessive is characterised by; (1) rarity of the trait (2) skipping of generations and
(3) consanguineous marriages. Figure 6.3 illustrates this.

Figure 11.12. A pedigree showing a pattern of inheritance consistent with a trait inherited as an
autosomal recessive. Source: Hayward.1992. Applied Genetics. Page 201.

11.5.2.3 Sex-linked traits

Sex-linked traits are carried by sex chromosomes (X or Y). Sex-linked (X-linked) are
characterised by: (1) Many more males than females being affected (2) If caused by a very rare
recessive gene, almost all observed cases would be males. (3) Usually none of the offspring of an
affected male will be affected (if his wife is unaffected) but all his daughters will carry the allele,
so half their sons, on average should be affected. Figure 6.4 illustrates this.

Figure 11.13. A pedigree showing the inheritance of an X-linked recessive trait. Source:
Hayward.1992. Applied Genetics. Page 201.
Some common human genetic diseases are shown in Table 11.1

Table 11.2. Some genetic diseases

Disease Type of inheritance Type of allele
Cystic fibrosis Autosomal Recessive
PKU Autosomal Recessive
Sickle cell anaemia Autosomal Recessive
Colour blindness X-linked Recessive
Haemophilia X-linked Recessive
Huntington’s disease Autosomal Dominant
Muscular dystrophy X-linked Recessive

11.6 Revision exercise

1. Compare and contrast prenatal screening based on amniocentesis and chorionic villus
sampling.

2. A woman with Turner syndrome is found to be colour-blind. Both her mother and father have
normal vision. How can this be explained?

3. Explain why only the daughters in Figure 1 express the trait. Under what circumstances could
sons inherit this trait?

Figure 12.14. A pedigree showing the inheritance of an unknown trait. Source:

Hayward.1992. Applied Genetics. Page 202.
4. Study Figure 2 below.

Figure 11.15 A pedigree illustrating the inheritance of an autosomal form of muscular

dystrophy which shows incomplete penetrance.
(a) Which individual first drew the geneticist’s attention to the family?
(b) Explain why E.1 and E.2 have not developed muscular dystrophy.
(c) Would you expect E.1 and E.2 to develop the disease?
(d) What advice would you give them about having children?

11.7 Summary

A chromosome mutation may be in number (due to missing or extra, chromosomal DNA), or in

structure (due to an irregular portion of chromosomal DNA). Structural mutation may be due to
deletion (deficiency), duplication, translocation or inversion. The major two types of mutation in
chromosome number are euploidy (changes in number of whole chromosome sets) and
aneuploidy (changes in number of individual chromosomes). Chromosomal mutations are
responsible for a number of human genetic diseases such as Down Syndrome, Klinefelter
Syndrome, cancers and sexual abnormalities. Methods such as amniocentesis, chorionic villi
sampling and ultra sound have been developed to detect genetic disorders in the unborn
children. Pedigree analysis helps estimate the probability of a couple having a child with a
genetic disorder.