Mutations are alterations in DNA sequences that can be caused by errors during DNA replication or DNA damage. They can occur at different levels, from single nucleotide changes to changes involving entire chromosomes. Mutations are important because they can cause genetic disorders and cancer, and provide the genetic variation that natural selection acts upon to drive evolution. The frequency of mutations is tightly regulated, as too many mutations could be harmful, while some level of variation is necessary for adaptation and survival. Gene expression is also tightly controlled, with most genes only being expressed when needed through transcriptional and translational regulation mechanisms like operons, promoters, operators, and repressors. This ensures efficient use of cellular resources.

1. Mutations

2.  A mutation is an alteration/damage to the nucleotide sequences of a
DNA molecule that is present in a relatively small number of
individuals.
 A change in DNA sequence that is present in at least 1% of the
population is a polymorphism.
 Both mutations and polymorphisms are transmissible even though
mutations are normally spoken of in the negative sense as against
polymorphisms.
 Mutations are important because they alter genes and are responsible
for inherited disorders and cancer. A study of mutations helps us
understand these conditions.
 Mutations are the source of phenotypic variation upon which natural
selection acts, where the favouring of any advantageous variants is the
driving force of evolution.
Importance of mutations

3.  The sequence of nuclear DNA is almost 99.9% identical between two
humans.
 This 0.1% small DNA sequence difference among individuals is
responsible for the genetically derived variability among humans.
 Many of such differences have little or no effect on the phenotype
whereas other differences are responsible for causing disease.
 Between these two extremes is the variation responsible for differences
in anatomy, physiology, dietary intolerances, therapeutic responses or
adverse reactions to medications, susceptibility to infections,
predisposition to cancer amongst others.
 These differences which favour a population under a particular stressor
serves as the driving force for evolution and our survival .
Genetic variation

4. Human mutations
 These are mutations that affect the number of chromosomes in the cell
arising from errors in chromosomes segregation during mitosis and
meiosis.
 A genomic mutation that deletes or duplicates an entire chromosome
alters the dosage and expression levels of hundreds or thousands of
genes. An example is the trisomy of chromosome 21 and its resulting
Down’s syndrome
 In most cases the results are so disastrous that foetuses are
spontaneously aborted even before the realization of pregnancy.
Genomic mutations

5.  These are changes involving large chromosomal regions either within the
same chromosome or to another chromosome. It can be a deletion,
translocation, or a duplication amongst others.
 These mutations alter the structure of individual chromosomes and can
either occur spontaneously or from abnormal segregation during meiosis.
These mutations can also affect hundreds of genes.
 These mutations are incompatible with survival or normal development so
they are not perpetuated from one generation to the next. They are
frequently seen in cancer cells.
Chromosome mutations

6.  These are changes in DNA sequences ranging from a single nucleotide
change to changes in thousands of base pairs.
 It differs from the other two in that the difference comparatively is on a
smaller scale.
 Even though these mutations may be small, they can have magnified
effects depending on the type of gene altered and the effect of alteration
on the gene
 Gene mutations can originate by either errors introduced during the
normal process of DNA replication or failure of the DNA repair system to
correct the error.
 In the latter case, some of the mutations are spontaneous whilst others
are induced by physical and chemical agents celled mutagens.
Gene mutations

7. Mutations occur at different frequencies in different cells. If it occurs in the
germline, the mutation is passed on to subsequent generations and they are
referred to as germline mutations e.g. thalidomide which was to help with
morning sickness was a teratogen that caused severe birth defects.
If however the mutation occurs a subset of cells of other tissues apart from
the germline, then they are somatic mutations or somatic mosaicism and can
not be transmitted to the next generation e.g. cancers.
Somatic and germline mutations

8. Types of gene mutations
Single nucleotide changes
1. Silent mutations – This often happens in the third codon and there may be
no change in the amino acid specified e.g. change from CGA to CGG does
not affect the protein because both code for arginine. This is due to the
degeneracy of the code.
2. Conservative mutations – The mutation results in a change of amino acid,
however the replacement and original amino acids both have similar
biochemical properties and the change doesn’t affect protein function
significantly e.g. leucine for valine
3. Missense mutations – Here the mutation results in one amino acid being
replaced by a different one. A change from CGA to CCA causes arginine to
be replaced by proline. This accounts for almost half of all mutations
reported to cause genetic disease in humans. The sickle cell trait is an
example. An adenine is replaced by a thymine causing a hydrophillic
glutamic acid to be replaced by a hydrophobic valine.

9. 4. Nonsense mutation – Formation of a stop codon from one that encodes an
amino acid. This results in premature termination of a polypeptide chain
e.g a change from CGA to UGA cause arginine to be replace by a stop
codon and the synthesis of the protein terminates here.
These mutations generally have no effect on transcription and constitutes
approximates 12% of all disease causing mutations e.g. One type of
thalassemia is caused by a nonsense mutation. Codon 17 of the β globin
chain changes from UGG to UGA, from tryptophan to stop codon.
Point mutations can either form or destroy a stop codon and
therefore the change in length of the protein.

10. Mutations that substitute a purine for a purine or a pyrimidine for a pyrimidine
are called transitions. The purine for a pyrimidine on the other hand is a
transversion. In nature, there is a higher frequency for transitions than
transversions.
Transitions and transversions
Mutations can be caused by the insertion or deletion of DNA sequences. In
deletions, one or more nucleotides are removed from the genetic sequence. If
the deletion is three or a multiple of three, it will remove a codon(s) and the
final protein product will be short and may or may not be operative.
Deletions
An insertion occurs when one or more nucleotides are added to the DNA. If it
does not generate a stop codon, then a protein with more amino acids could
be produced. Insertion or deletion of one or two nucleotides generates a
frameshift mutation which alters the reading frame.
Insertion

11. Deletions or insertions that are not multiples of three results in frame-shift
mutations. The reading frame shifts at the point where the insertion or
deletion begins. Beyond that, subsequent codons are read in the new context
until a termination codon is reached. The amino acid sequence and therefore
protein translated differs from the normal protein.
Frame-shift mutations
In fragile X syndrome and Huntington disease, there are trinucleotide repeats
(repetitive sequences in untranslated and coding regions respectively) with
detrimental outcome not because of altered amino acid sequence but rather
loss of protein expression.
Fragile X syndrome is the most common form of inherited mental retardation
(1/4000 in men and 1/8000 in women), second to Downs. Normal individuals
have about 60 CGG repeats in the FMR1 gene, but fragile X individuals have
over 230 repeats.
In Huntington, the mutated gene expresses a protein in the brain called
huntingtin from CAG repeats. Normal humans average 18 repeats whilst
diseased individuals have 40 or more repeats. Children show symptoms early.
Triple nucleotide repeats

12. The nature of the genetic code is such that frameshift mutations lead to
termination codon within a small number of codons. This characteristic may
have evolved to protect cells from making long nonsense proteins .
DNA sequence Amino acid
sequence
Type of mutation
ATG CAG GTG ACC TCA GTG M Q V T S V None
ATG CAG GTT ACC TCA GTG M Q V T S V Silent
ATG CAG UTG ACC TCA GTG M Q L T S V Conservative
ATG CCG GTG ACC TCA GTG M P V T S V Non-conservative
/missense
ATG CAG GTG ACC TGA GTG M Q V T S terminate Nonsense
ATG CAG GTG AAC CTC AGT G M Q V N L S Frame-shift
Types of point mutations

13. Mutation nomenclature
a. The position of a mutation is designated as being in either genomic or in
cDNA sequence by the prefix g. or c., respectively.
b. The A of the ATG start codon is designated as +1. Upstream is -1, there is
no zero.
c. A nucleotide change is noted first by the original base, the nucleotide
number of that base, a greater than (>) symbol, and the new nucleotide at
that position e.g. c. G1444>A.
d. Small deletions are indicated by the term “del” written after the nucleotide
numbers deleted (1524-1526del).
e. Small insertions also indicated by the term “ins” after the two nucloetides
between which the insertion occurred. C1277 – 1278insTATC.
f. If a missense mutation is described at the level of a protein, using
methionine as 1, the missense mutation is written as Glu6Val (E6V) as in
sickle cell. A stop codon is written as Gln39X (Q39X).

14. One code for glutamate is GAG and the code for valine is GUG. Assuming this
mutation is in sickle cell, what kind of mutation is it.
a. Transversion
b. Transition
c. Nonsense mutation
d. Insertion
Question 1
From the normal and mutants mRNA sequence denoting Arg-Ile-Ser-Tyr-Gly-
Pro-Asp below
5’-CGTATATCCTATGGCCCTGACCCA-3’ …….. Normal
5’-CGTATATCTATCCTATGGCCCTGAC-3’ …….. Mutant
a. The codon for Arg is CGA
b. The mutant protein will be shorter than the normal protein
c. The mutant gene has a insertion and thus a frameshift mutation
d. The mutant gene has a point mutation

15. What type of mutation is involved here. What possible nucleotides are
involved
a. Missense mutation
b. Nonsense
c. Silent
d. Repeat expansion
Question 3
Phe Thr Val Tyr Leu
TTT ACA GTT TAT CTC
Phe Thr Val STOP
TTT ACA GTT

16. Addition of multiple CAG repeats in huntington protein leading to
polyglutamine repeats. Aggregation of these repeats in neural tissue leads to
atrophy of the brain. Patients manifest with involuntary movements,
depression and cognitive impairment.
Huntington’s disease
Most viruses including the Flu virus transfers the 7-methyl G cap from host
cell mRNA to viral mRNA to increase its stability and its translation efficiency.
mRNA cap
The mutation is AF508. Deletion of the codon for phenylalanine at position 508
of the cystic fibrosis transmembrane conductance regulator (CFTR) results in
absence of the protein. The result is thickening of airway secretions and
patients suffer from recurrent pulmonary infections which are life threatening.
Cystic fibrosis
Of interest

17. No wonder Ghana is in deep shit

18. Regulation of gene expression

19.  This is simply the generation of a protein or RNA product from a particular
gene. At any given time only a fraction of the genes in a cell are expressed
meaning there must be some sort of regulation control.
 By regulating the activity of their genes, cells express the right genes at
the right time during development and differentiation.
 This control also enables cells conserve fuel since transcription and
protein synthesis are energy consuming.
 However, there are genes expressed all the time in almost all cells. These
are constitutive or housekeeping genes. RNA polymerases, DNA repair
enzymes, glycolytic enzymes, ribosomal proteins etc.
 Other genes are only expressed when needed and they are inducible
genes. Such expression is regulated by environmental, developmental or
metabolic signals.
Gene expression
Thus gene expression depends on the type of cell and chemical
signals from the environment

20. When and how often should gene be expressed
 For an efficient organism like bacteria the “when” is if the protein
product is required to perform a function.
 “How much” will depend on the substrates available for utilization by
the bacteria.
 Therefore intricate molecular mechanisms must tightly control the
expression of various genes throughout the life of a cell.
 Two possible ways of control are transcriptional control – when mRNA
molecules are being transcribed from DNA templates and translational
control – controlling the rate of synthesis of proteins from the
transcribed mRNA.
 Since it makes sense for the cell not to make more RNA molecules
than it needs, initial attention focused on transcriptional control.

21. However, in the rapidly changing microenvironment of the bacteria,
enzymes needed at one moment become useless soon afterwards and
enzymes not needed a few minutes ago may be suddenly required.
How does the bacteria tackle this problem

22. Inducers promote the binding of RNA polymerase
 To synthesize all enzymes irrespective of substrate will be a complete
waste of resources.
 In reality, bacteria make mRNA molecules only when their genes
receives signals from outside to go into action.
 One example of a signal is the inducer lactose which induces the
enzyme β-galactosidase in E. coli. (this enzyme splits lactose to
glucose, which is a utilizable food source and galactose.
 The presence of lactose increases the rate at which RNA polymerase
binds to the 5’ end of the gene to initiate the synthesis of β-
galactosidase mRNA.
 Inducers are thus examples of positive control as they promote gene
action.
Humans express about 10,000 – 15,000 genes from the
30,000 at any one time

23. Bacterial genes with related functions are
organized into operons
 In E. coli, there are two other genes in addition to β-galactosidase involved
in lactose metabolism.
 One gene encode lactose permease, which facilitate the entry of lactose into
the bacterial cell and the other is thiogalactosidase transacetylase which is
thought to remove lactose-like compounds that β-galactosidase can not
split into useful metabolites.
 The three genes encoding these enzymes are lacZ, lacY and lacA
respectively.
 Since all these genes are on the same mRNA, when lactose is added relative
amounts of all three proteins are synthesized.
 The collection of adjacent genes that are transcribed together with their
control region is called an operon.
 Genes in an operon are coordinately expressed i.e. they are either all turned
on or all turned off.

24. Promoters, operators and repressors
 The site where RNA polymerase binds in order to initiate transcription of an
operon is called the promoter, which is a non coding sequence upstream
the transcription start point.
 In E. coli, two highly conserved sets of nucleotide blocks upstream the
initial codon make up its promoter.
 The initial step is believed to be the recognition and binding of the RNA
polymerase to this region. This then allows transcription to start at the +1.
There is thus a specific place for RNA polymerase binding.
 However, the binding of RNA polymerase is controlled through repressors,
which are regulatory proteins that slow down or prevent RNA binding.
 In our example LacI gene encodes a repressor that regulates the lac
operon.
 The repressor is encoded by the regulatory gene which is part of the
operon. Its product, the repressor protein binds to a region of the operon
call the operator.

25. Mechanism of repression (active repressor)

26. Mechanism of an inducer

27.  Here the synthesized repressor actively binds to the operator and
prevents RNA polymerase binding.
 If lactose becomes available, the three enzymes required for lactose
metabolism needs to be synthesized.
 A metabolite of lactose - allolactose serves as the inducer and binds to
and inactivates the repressor.
 RNA polymerase can now bind to the promoter and transcribe the
polycistronic mRNA for the three genes.
 Upon removal of allolactose, the repressor regains its ability to bind to
the operator DNA, dislodges RNA polymerase and terminate the
transcription of lac mRNA.
The lac operon is an inducible operon
It is off till an inducer combines with the repressor

28. Lactose is not essential so the operon doesn’t need to be
on continuously
CAP lacP

29. Lactose is converted to allolactose, which serves as the
inducer

30. Co-repressors – tryptophan operon
 In co-repression, the repressor is inactive with respect to binding the
operator till a small molecule called the co-repressor (nutrient or
metabolite), binds to the repressor and activates it.
 The activated repressor-co-repressor complex can now bind to the
operator and prevents binding of RNA polymerase and thus gene
transcription.
 The tryptophan operon which encodes 5 enzymes for synthesis of
tryptophan is an example of a repressible operon.
 Under normal conditions, the trp repressor which is the product of a
regulatory gene called trpR is synthesized in an inactive form with little
affinity for the operator.
 Only when tryptophan binds does it become active and binds to the
operator.
 Tryptophan thus serves as a co-repressor that binds to the inactive
repressor, changing its conformation and allowing it to bind to the
operator to inhibit transcription.

31. Tryptophan is essential so the operon is on for RNA
polymerase to bind and transcribe

32. When tryptophan is in excess, it binds the repressor, making it
active so it can attach to the operator
The trp operon is a repressible operon

33. If lactose permease (which increases lactose entry into the cell) is induced
by lactose, then how does lactose initially gets into the cell to induce these
enzymes?
Question
Regulatory proteins are expressed continuously although at a very low rate.
Thus a small amount of the permease exists even in the absence of lactose
and allows a few molecules of lactose to enter the cell. These lactose are
metabolized to allolactose which begins the process of inducing the
operon. As the amount of the permease increases, more lactose can be
transported into the cell.
Answer

34. The lac operon is subject to further regulation in the presence
of glucose
The presence of glucose
 Lactose is broken down to glucose and galactose.
 Glucose can be utilized by the bacteria but galactose will have to be
converted to glucose for usage.
 So, what happens if the bacteria finds itself in an environment that has
both glucose and lactose.
 Being that efficient, the bacteria will have no use for the lactose enzymes,
thus the presence of glucose actually prevents the activation of the lac
operon

35.  Bacteria can also regulate transcription by activating regulatory
proteins that bind to a regulatory region of the promoter to ensure RNA
polymerase binding.
 For example, transcription of the lac operon in the presence of lactose
can be induced by allolactose only if glucose is absent.
 In glucose absence, E. coli senses and increases the interaction of a
regulatory protein with cyclic AMP (cAMP).
 cAMP binds and activates catabolite activator protein (CAP) to form the
cAMP-CAP complex (cAMP-CAP), which then binds to the regulatory
region and promotes binding of the RNA polymerase to the lac operon.
 When glucose is present, cAMP levels decrease and there is no
formation of the complex to bind to the operon and transcription is
inhibited.
Catabolite repression (Positive gene regulation)
When glucose and lactose are present, the bacteria
preferentially uses glucose

36. Lactose present and glucose absent
Absence of glucose increases cAMP so cAMP-CAP complex forms
The lac RNA polymerase will only bind and transcribe when
cAMP-CAP complex binds the promoter region

37. Lactose and glucose present
Even though allolactose will inactivate the repressor, without the
c-AMP-CAP complex, RNA polymerase is prevented from
binding
Presence of glucose reduces cAMP so no cAMP-CAP complex

38. Which of the following double-stranded sequences shows perfect
dyad symmetry (same sequence of bases on both strands)
a. GAACTGCTAGTCGC
b. GGCATCGCGATGCC
c. TAATCGGAACCAAT
d. GCAGATTTTAGACG
e. TGACCGGTGACCGG
Question 1
b

39. In E. coli, under high lactose, high glucose conditions, which of the
following could lead to maximal transcription activation of the lac
operon.
a. A negative mutation in the lac I gene (which encodes the
repressor)
b. A mutation in the CAP binding site leading to reduced CAP
binding
c. A mutation in the operator sequence
d. A mutation leading to enhanced cAMP levels
e. A mutation leading to lower binding of repressor.
Question 2
d

40. A mutation in the I (repressor) gene in a strain of E. coli resulted in
the inability to synthesize any of the proteins of the lac operon.
Which of the following provides a rational explanation.
a. The repressor has lost its affinity for the inducer
b. The repressor has lost its affinity for the operator
c. A trans acting factor can no longer bind to the promoter
d. The CAP protein is no longer made
e. Lactose feedback inhibition becomes constitutive
Question 3
a

41. Which of the following explains why several different proteins can be
synthesized from a typical prokaryotic mRNA
a. Any of the three reading frames can be used
b. There is redundancy in the choice of codon/tRNA interactions
c. The gene contains several operator sequences from which to
initiate translation
d. Alternative splicing events are commonly found
e. Many RNAs are organized in a series of consecutive
translational cistrons
Question 4
e

43. Regulation in eukaryotes
 Regulation in multicellular eukaryotes are more complex.
 During development, different sets of genes are turned on to produce
different protein groups capable of performing distinct functions.
 Beyond that, certain cells continue to differentiate e.g. cells producing
antibodies in response to infection, cells that renew the population of
red blood cells.
 Eukaryotes have three RNA polymerases so their regulation will
definitely be different from prokaryotes.
 Eukaryotic gene regulatory proteins can control gene expression from a
distance at sites called enhancers
These differences show that gene expression in eukaryotes is
complex and therefore regulation must occur at multiple levels.

44. Differences in regulation
 Eukaryotic DNA is organized into the nucleosomes and solenoids of
chromatin and must be in an active structure in order to be expressed.
 There are no operons present and genes encoding proteins that function
together can even be located on different chromosomes.
 Each gene needs its promoter, as transcription and translation are
separated by compartments and time.
Regulation can thus occur at
a) Chromosome modeling and gene arrangement
b) Transcription initiation through factors affecting RNA polymerase
binding
c) Processing of transcripts
d) Stability of the mRNA and initiation of translation.
e) Post translation

45. Control points for regulation in eukaryotes

46. Control points for regulation in eukaryotes

47. Model for action of enhancers and
transcriptional activators
This enhancer though a
distance away has three
essential binding sites for
activator proteins
DNA bending proteins brings
bound activators closer to the
promoters
The activators bind to the
mediator proteins and
general transcription factors
to form the initiation
complex

48. Availability of genes for transcription
 A typical nucleus contains chromatin that is condensed
(heterochromatin) or diffused (euchromatin). The genes in the
heterochromatin are inactive whilst those in the euchromatin produce
mRNA.
 Since DNA is packed tightly into nucleosomes, unless there is a change
of state (chromatin remodeling), transcription can not be initiated. So
chromatin remodeling displaces the nucleosomes from specific DNA
sequences so that transcription can be initiated.
Chromatin remodeling
 Cytosine residues can be methylated in GC-rich sequences near or in
the promoter region. Methylated genes are less readily transcribed and
is a mechanism for regulating gene expression during differentiation.
Methylation of DNA

49. 1. Nearly all eukaryotic genes have regulatory DNA sequences (10-1000
nucleotides upstream) that are used to switch the gene off or on.
2. These DNA sequences are recognized by gene regulatory proteins or
transcription factors that bind DNA sequences directly. They include
activators, repressors, inducers and nuclear receptors amongst others.
3. Gene regulatory proteins contain a variety of DNA binding motifs that
binds to a specific sequence of nucleotides in DNA because a portion of
their surface fits tightly into the double helix at that region.
4. Binding is between the α-helix of the protein and the major groove of
the DNA. The bonds formed are hydrogen, ionic, and hydrophobic
interactions
5. Due to the large number of protein-DNA contacts, the interaction is
specific and strong as different proteins recognizing different DNA sites
Gene-specific regulatory proteins/transcription factors
Regulation at transcription
Control of the transcription complex assembly

50. Posttranscriptional processing
Regulation can occur during the processing of the pre-mRNA to the mature mRNA.
Different combinations or rearrangement of the exons produces different
proteins from the same gene.
Alternative splicing

51. RNA editing of apolipoprotein B
Although the sequence of the gene and primary transcript will be the same,
bases can be altered by substitution, addition or deletion so that the mature
mRNA differs in different tissues.
The synthesis of apolipoprotein B100 in the liver and apolipoprotein B48 in
the intestines is a good example.
RNA editing

52. Regulation at translation
a) At the level of protein synthesis initiation by the phosphorylation of the
eukaryotic initiation factors (eIFs).
In addition to stimulating degradation of mRNA, interferon also
causes the phosphorylation of eIF2 rendering it inactive. This is
the main mechanism by which interferon prevents the synthesis
of viral proteins.
Regulation of translation
b) A second mechanism is by allowing translation to proceed only when
there is demand for the protein product.
Ferritin, the protein involved in storage of iron within cells (16-
18% of total body iron) is synthesized when iron levels increase.
Its mRNA has an iron response element (IRE) at its 5’ end and can
bind to a regulatory protein called IRE binding protein (IRE-BP).
When IRE-BP does not contain bound iron, it binds to IRE and
prevents translation. When iron levels increase, the IRE-BP can
now bind to iron, releasing its hold on IRE to allow translation.

53. Translational regulation of ferritin synthesis

54. Transport and stability of mRNA
1. mRNAs with long half lives can generate a greater amount of protein
than those with shorter half lives.
2. To prevent degradation in the nucleus or cytoplasm before it is
translated, mature mRNA has the 3’ poly(A) tail.
3. An example of the role of mRNA degradation in control of translation is
the transferrin receptor mRNA.
4. The transferrin receptor protein permits cells to make transferrin, the
protein that transports iron in the blood.
5. When iron levels are low, the rate of synthesis of the transferrin receptor
increases, thus enabling cells take up more iron.
6. The synthesis of the transferrin receptor also depend on IRE and IRE-
BP. However in this case, the IRE are at the 3’end.
7. When iron is low, the IRE-BP binds to the IRE and prevent mRNA
degradation so more iron can be taken in by the cells.
8. When iron level are high IRE-BP releases IRE and the mRNA is
degraded.

55. Transferrin receptor synthesis