2. Viral genetics
• A virus is a microscopic obligate intracellular
infectious parasite that can infect cells and
replicate in them but does not exhibit living
functions on its own.
–Infectious Agents Other Than Viruses
• Prions are infectious protein particles
responsible for degeneration of the nervous
system, mainly in humans and sheep.
• Viroids are naked snippets or pieces of RNA
(no protein coat) that cause plant diseases.
3. Structure of viruses
• Made up of nucleic acid core (genetic material)
• Surrounded by protein coat called capsid,
protect viruses from external environment
• The protein sub-units that constitute the capsid
are called capsomeres.
• Proteins making up the capsid is determine by
the kind of nucleic acid
• Some viruses are also further surrounded by
lipoprotein envelope made up of protein, lipids,
carbohydrates/glycoprotein molecules
• Viruses are extremely small and vary in size.
4. Characteristics of viruses
• Are acellular (akaryotic)- lack true membrane and organelles
• Cannot carry out metabolic activities independently,
• no respiration, protein synthesis, cannot reproduce on it’s own
• Because it lacks many of the enzymes and structures necessary
for reproduction, protein synthesis and ATP generation.
• Contain DNA or RNA which may be ssDNA/ dsDNA/ ssRNA/
dsRNA
• Crystallizes
• Vary in shape
5. Shape of Viruses
• Helical/icosahedral viruses: rod-shaped with
capsid protein winding around the core in a
helix.
• Polyhedral/isomeric: Capsid appears spherical
eg HIV
• Helical + Polyhedral (complex virus): made up
of head, neck, tail, base plate and tail fibres eg
bacteriophages
9. Host range
• A virus can only infect and reproduce within
certain living host cells.
• Viruses identify their hosts by specific
receptor molecules on the outside of the
host cell.
• Some viruses have broad host ranges and can
infect many different types of cells.
• Other viruses, have extremely narrow host
ranges and can infect only very specific cells.
10. Host specificity
• Some viruses have high host specificity. Plant
viruses do not infect animals and vice versa;
bacteriophages infect only bacteria
• At the highest specificity, certain viruses infect
only specific cells eg Human Immunodeficiency
Virus (HIV) infects only CD4 Lymphocyte cells
• Hepatitis virus infect only liver cells
• Rabies virus infects the brain cells
11. Life cycle of viruses
• There are modified life cycles depending on
the type of virus and host.
Bacteriophage is a virus that infects bacteria: The
phage T4 and the phage lambda, for example, both
infect E. coli.
viruses can multiply by two alternative
mechanisms
• Lytic cycle
• Lysogenic cycle
12. Lytic cycle
• Involves 5 stages:
• 1. Attachment
o Viruses attaches to a receptor glycoprotein on the cell's
surface or by simple mechanical force.
o Phage T4 uses tail fibers to attach to complementary
receptor sites on cell wall of E. coli.
o Weak chemical bonds form between the
attachment and receptor sites, adhering the virus to
the host.
14. Entry pathways:
Virus can fuse either directly to the plasma membrane (receptor-mediated fusion) or
by endocytosis. Entry routes depends on the type of virus. In fusion with the plasma
membrane, the virus binds to protein receptors in the cell membrane. The receptor
proteins induce a conformational change in the viral fusion protein, leading to fusion.
For virus that is triggered within an endosome, the endosome’s acidic conditions
induce fusion. In either case, the viral genome passes through a fusion pore into the
cytosol and to initiate infection.
15. Cont’d
• 2. Penetration
• Virus injects its DNA into E. coli/bacterium by
releasing the enzyme, phage lysozyme, which breaks
down a portion of the cell wall.
• The bacteriophage contracts it’s tail sheath driving
the tail core into E. coli.
• Viral DNA is injected and passes through the core
into host cell while the capsid remains outside
• NB: Many viruses that infect animal cells enter the
host cell intact with the aid of vectors. In doing this,
the cell is infested and can also be targeted by the
immune system.
16. Cont’d
• 3. Biosynthesis
• Virus DNA hijacks gene expression mechanism of the host cell genome
• Virus direct synthesis of enzymes to degrade host cell DNA and interrupt
transcription and translation
• Virus uses host nucleotides to replicate its DNA, transcription into mRNA
and host ribosomes to synthesize its enzymes and proteins.
• During this time no complete phages are found in the host and is called
the eclipse period.
NB: In retroviruses (which inject an RNA strand), a unique enzyme called
reverse transcriptase transcribes the viral RNA into DNA, which is then
transcribed into mRNA.
17. Cont’d
• 4. Maturation/Assemblage/packaging
• Spontaneous assembly of capsids and packaging of DNA
inside the head.
• Tails fibers join the complex.
5. Release
• Lysozyme produced by the phage destroy the host cell
membrane and cell wall to cause rapid cell lysis and
release viruses at once. The host cell dies.
• Burst time is the time from attachment of a virus to
lyses and release of the new phage particles, 20-40 min.
• Burst size is the number of viruses released from the
cell at the burst time, 50-200 viruses
18. Lysogenic Life Cycle
• When a virus enters a lysogenic cycle, its DNA is
inserted and recombines with Host DNA.
• The inserted phage DNA is called a prophage.
• Repressor proteins, which are products of the
prophage, prevent transcription of the other
prophage genes.
• As a result, the synthesis and release of new phages is
repressed.
• Every time the bacterial chromosome is replicated,
prophage DNA is replicated, as well. The prophage can
remain latent in the bacterial chromosome for many
generations.
• A spontaneous event (UV radiation, X-ray) at any time
may cause the virus to break out of its latent state and
enter the lytic cycle.
20. Structure of coronavirus
A. Whole Structure B. Cross section structure
https://en.wikipedia.org/wiki/Coronavirus#/media/
File:3D_medical_animation_coronavirus_structure.jpg
21. Recombination
• Viral recombination occurs when viruses of two different
parent strains coinfect the same host cell and interact
during replication to generate virus progeny that have
some genes from both parents.
• RNA viruses are capable of genetic recombination when at
least two viral genomes are present in the same host cell.
• RNA recombination appears to be a major driving force in
determining genome architecture and the course of viral
evolution.
• Recombination in RNA viruses appears to be an adaptation
for coping with genome damage.
• The resulting recombinant viruses may sometimes cause an
outbreak of infection in humans.
22. Advantage of recombination
• Virus genome can evolve by recombination.
These recombination events can be of
evolutionary advantage for the virus when it
helps to evade host immune defenses, for
example by changing surface protein
antigenicity.
23. Questions
1. With the aid of annotated diagrams describe
lytic and lysogenic cycles of viruses.
2. Outline structural feature of viruses that are
controlled by genes.
3. State the function of repressor proteins in
the lysogenic cycle. Ans: The receptor proteins induce a
conformational change in the viral fusion protein, leading to fusion of the virus to the
host’s cell membrane.
4. Briefly explain genetic recombination with
reference to viruses.
5. State and explain the five stages of lytic
cycle.
24. The Central Dogma theory
• Transcription of DNA to RNA to protein.
• ie DNA→RNA → Protein:
• This dogma forms the backbone of molecular biology
and is represented by four major stages.
• 1.DNA replication: The DNA replicates its
information in a process that involves many
enzymes:
• 2. The DNA codes for the production of messenger
RNA (mRNA) known as transcription.
25. Cont’d
• 3. In eukaryotic cells, the mRNA is processed
(essentially by splicing) and migrates from the
nucleus to the cytoplasm.
• 4. mRNA carries coded information to
ribosomes. The ribosomes "read" this
information and use it for protein synthesis.
This process is called translation.
• Proteins do not code for the production of
protein, RNA or DNA. They are involved in
almost all biological activities, structural or
enzymatic.
28. The Structure & Complexity of Virus
Genomes
• The composition & structure of virus genomes
(i.e. the genetic material which encodes the
genetic information of the virus) is more
varied than those of bacteria, plant or animal.
• Composition - DNA or RNA, single-stranded or
double-stranded, linear or circular.
• The nucleic acid may be non-segmented,
segmented and multipartite
• Size & number of segments varies.
30. Differences between enhancers
and promoter
• An enhancer is a piece of DNA that enhances gene
transcription by bringing transcription factors close to
promotors but a promoter is a piece of DNA which acts to
initiate or start gene transcription, determines where
transcription occurs and direction.
• An enhancer binds with transcription factors while a promoter
binds with transcription factors and RNA polymerase enzyme.
• An enhancer can be upstream or downstream from the site
where transcription is initiated while a promoter is always
upstream from the site where transcription is initiated.
• An enhancer does not need to close to transcription initiation
site but promoter need to be close to the transcription
intiation site.
31. Cont’d
• RNA viruses generally have very high mutation
rates compared to DNA viruses,because viral
RNA polymerases lack the proofreading ability
of DNA polymerases.
• Hence they are copied less accurately, driving
RNA viruses towards smaller genomes.
• This is one reason why it is difficult to make
effective vaccines to prevent diseases caused
by RNA viruses.
32. Cont’d
• Single-stranded virus genomes may be:
• Positive (+)sense, i.e. of the same
polarity (nucleotide sequence) as mRNA
• Negative (-)sense
• Ambisense - a mixture of the two.
33. Positive-Strand RNA Viruses:
• Positive (+)sense RNA are infectious when the purified RNA
is applied to cells in the absence of any virus proteins.
• Infection of cells caused by nucleic acid alone is referred to
as transfection:
• This is because (+)sense RNA is essentially mRNA & the first
event in a normally-infected cell is to translate the RNA to
make the virus proteins responsible for genome replication.
• In most cases, virus genomes which are composed of double-
stranded DNA are also infectious. The events which occur
here are a little more complex, since the virus genome must
first be transcribed by host polymerases to produce mRNA.
• Using these techniques, virus can be rescued from cloned
genomes, including those which have been manipulated in
vitro.
35. Cont’d
• Purified (+)sense vRNA is directly infectious when
applied to susceptible host cells in the absence of any
virus proteins. Both ends of (+)stranded eukaryotic
virus genomes are often modified
• There is a methylated untranslated region (UTR) at
the 5' end of the genome.
• A shorter polyadenylated UTR at the 3' end.
• These regions are functionally important in virus
replication & are thus conserved in spite of the
pressure to reduce genome size.
• These signals allow vRNA to be recognised by host cells
& to function as mRNA.
36. Negative-Strand RNA Viruses:
• Negative-sense RNA genomes are not infectious as
purified RNA because they lack RNA polymerase.
• Purified negative-sense genomes cannot be directly
translated & are not replicated in the absence of the
virus polymerase.
• NB: The first event when the virus genome enters the
cell is that the (-)sense genome is copied by the
polymerase, forming either (+)sense transcripts which
are used directly as mRNA, or a double-stranded
molecule known either as the replicative intermediate
(RI) or replicative form (RF), which serves as a template
for further rounds of mRNA synthesis.
37. Segmented & Multipartite Virus
Genomes
• Segmented virus genomes are those which are
divided into two or more physically separate
molecules of genetic material packaged into a
single virus particle.
• Multipartite genomes are those which are
segmented and each genome segment is
packaged into a separate virus particle. These
discrete particles are structurally similar & may
contain the same component proteins, but often
differ in size depending on the length of the
genome segment packaged.
40. Cont’d
• There are many examples of segmented virus
genomes, including many human, animal & plant
pathogens such as orthomyxoviruses, reoviruses
& bunyaviruses.
• There are rather fewer examples of multipartite
viruses, all of which infect plants. These include:
• bipartite viruses (which have two genome
segments/virus particles)
• tripartite viruses (three genome segments/virus
particles)
41. Cont’d
• Separating the genome segments into different particles
removes the requirement for accurate sorting, but introduces
a new problem in that all of the discrete virus particles must
be taken up by a single host cell to establish a productive
infection. This is perhaps the reason why multipartite viruses
are only found in plants.
• Many of the sources of infection by plant viruses, such as
inoculation by sap-sucking insects or after physical damage to
tissues, results in a large input of infectious virus particles,
providing the opportunity for infection of an initial cell by
more than one particle.
42. Cont’d
• Since viruses are obligate intracellular
parasites, only able to replicate inside the
appropriate host cells, the genetic code
employed by the virus must match or be
recognized by the host organism.
• Similarly, the control signals which direct the
expression of virus genes must be appropriate
to the host.
43. Cont’d
• There are a number of virus groups which
have double-stranded DNA genomes of
considerable size & complexity.
• In many respects, these viruses are genetically
very similar to the host cells which they infect.
Two examples of such viruses are the
adenovirus & herpesvirus families:
44. Cot’d
• Viruses with negative-sense RNA genomes are
a little more diverse than positive-stranded
viruses. Possibly because of the difficulties of
expression, they tend to have larger genomes
encoding more genetic information. Because
of this, segmentation is a common though not
universal feature of such viruses.
45. MBB302: Microbial Genetics- Part II
Cloning
• In Biology, cloning is the process of producing similar
populations of genetically identical individuals that
occurs in nature when organisms such as bacteria,
insects and plants reproduce asexually.
• Cloning in biotechnology refers to processes used to
create copies of DNA fragments/gene (
molecular cloning), cells (cell cloning), or organisms.
• The term cloning also refers to the production of
multiple copies of a product such as digital media or
software.
46. Molecular cloning
• Molecular cloning refers to the process of making multiple
copies of genetically identical molecules- DNA/genes.
Uses of molecular cloning
• Cloning is commonly used to amplify DNA fragments
containing whole genes,
• To amplify any DNA sequence such as promoters, non-
coding sequences and randomly fragmented DNA.
• Used in biological experiments and practical applications
ranging from genetic fingerprinting to large scale protein
production.
47. Cloning vector
• cloning vectors - small piece of DNA into
which a foreign DNA fragment can be inserted
that allow protein expression, tagging, single
stranded RNA and DNA production and other
manipulations.
• DNA clone = A section of DNA that has been
inserted into a vector molecule and then
replicated in a host cell to form many copies.
48. Requirements for a cloning vector
• a) Should be capable of replicating in host cell
• b) Should have convenient RE sites for inserting DNA of interest
• c) Should have a selectable marker to indicate which host cells
received recombinant DNA molecule
• d) Should be small and easy to isolate
Eg. 1. Bacterial plasmids as cloning vector
• Small, circular DNA molecules that are separate from host cell
chromosome.
• They replicate independently of the bacterial chromosome.
• Useful for cloning DNA inserts less than 20 kb (kilobase pairs).
Inserts larger than 20 kb are lost easily in the bacterial cell.
49. Cloning vectors
Eg. 2. Cosmids- are hybrids of phages and
plasmids that can carry DNA fragments up to
45 kb. They can replicate like plasmids but can
be packaged like phage lambda.
Eg. 3. Expression vectors- are vectors that carry
host signals that facilitate the transcription
and translation of an inserted gene. They are
very useful for expressing eukaryotic genes in
bacteria.
50. Cloning vectors
Eg.4. Yeast artificial chromosomes (YACS) - are yeast
vectors that have been engineered to contain a
centromere, telomere, origin of replication, and a
selectable marker.
• Can carry up to 1,000 kb of DNA.
• They are useful for cloning eukaryotic genes that
contain introns since they are maintained in yeast
(a eukaryote).
• Also, eukaryotic genes are more easily expressed
in eukaryotic host such as yeast.
51. Cloning vectors
5. Bacterial artificial chromosomes (BACS) - are
bacterial plasmids derived by engineering from
the F plasmid. They are capable of carrying up to
300 kb of DNA.
6. Bacteriophage lambda (45 kb) contains a central
region of 15 kb that is not required for replication
or formation of progeny phage in E. coli. Thus,
lambda can be used as a cloning vector by
replacing the central 15 kb with 10-15 kb of
foreign DNA.
52. Expression vector
• An expression vector/ expression construct, is usually a
plasmid or virus designed for protein expression in cells.
• The vector is used to introduce a specific gene into a target
cell, and can commandeer the cell's mechanism for protein
synthesis to produce the protein encoded by the gene.
• The plasmid is engineered to contain regulatory sequences
that act as enhancer and promoter regions and lead to
efficient transcription of the gene carried on the expression
vector. The goal of a well-designed expression vector is the
production of significant amount of stable messenger RNA,
hence proteins.
54. Analysis of cloned gene
1. Gel electrophoresis – DNA fragments of
different sizes can be separated by an
electrical field applied to a “gel”. The
negatively charged DNA migrates away from
the negative electrode and to the positive
electrode. The smaller the fragment the faster
it migrates.
57. Analysis of cloned gene
2. Southern Blotting- allows the detection of a gene of
interest by probing DNA fragments that have been
separated by electrophoresis.
Transfer DNA fragments to a support matrix , a
nitrocellulose membrane and hybridized to a radioactive
"probe“ for example, 32
P-labeled DNA. DNA-DNA
hybridization. This technique is called Southern blotting
3. Northern Blotting-probe RNA on a gel with a DNA or RNA
probe RNA-DNA or RNA-RNA hybridization
4. Western Blotting - probe proteins on a gel with an antibody
5. DNA sequencing of a gene
6. Restriction enzyme mapping
7. PCR (polymerase chain reaction) – Allows the isolation of a
specific segment of DNA from a small DNA (or cell sample)
using DNA primers at the ends of the segment of interest.
58. Polymerase Chain Reaction
• A very quick, easy, automated method used to
make copies (clone) of a specific segment of DNA
• The discovery of thermostable DNA polymerases,
such as Taq Polymerase, made it possible to clone
DNA segment in the laboratory using PCR.
• Primers specific to a particular region of DNA, on
either side of the gene of interest, are used in PCR
reaction to amplify or multiply segment of DNA
• Replication is stopped and started repetitively,
generating millions of copies of that gene. These
copies can then be separated and purified using gel
electrophoresis.
59. Components of PCR mixture
• 10x PCR Reaction Buffer
• MgCl2
• dNTPs (dATP, dGTP, dTTP and dCTP)
• Forward Primer
• Reverse Primer
• Target DNA/Genomic DNA
• Taq. Polymerase - Polymerase enzyme
Sterile Distilled Water
60. PCR reaction conditions
Instrument used for PCR - Thermocycler
• The reaction conditions of a PCR amplification are
composed total number of cycles to be run,
temperature and duration of each step in those cycles.
• Generally 25 to 35 cycles is the standard for a PCR
reaction
• 1. Initial Denaturation at 94/95 oC for 5 min.
• 2. DNA denaturation step: 94 oC or 95 oC for 30 Sec.
• 3. Primer annealing step- depends on melting
temperature of the primers used usually 50 oC – 60 oC
for 30 Sec. – 45 Sec.
• 4. Polymerase extension step- 72 oC for 2 min
• 5. Final extension step- 72 oC for 5 min.
61. PCR cont’d
• A convenient shorthand way
• of representing a complete set of reaction conditions
is:
• 94o
C5:00
[94 o
C0:30
; 55 o
C0:45
; 72 o
C2:00
]35; 72 o
C7:00
• which means an initial denaturing step of five minutes
at 94oC followed by 35 cycles of denaturation 94oC for
30 seconds, annealing 55 oC for 30 seconds and
extension 72oC for two minutes and then a final
extension at 72oC for seven minutes.
63. Strategies used to Genetically Engineer Bacteria
An overview of how bacterial plasmids are used to clone genes
1. Isolate the gene of interest (e.g. insulin gene)
2. Insert the gene of interest into a bacterial R-
plasmid
• R-plasmids are circular DNA molecules found in
some bacteria that provide resistance to up to
10 different antibiotics
3. Place the transgenic plasmid into bacterial
cells
• Plasmid DNA reproduces each time the bacteria reproduce
4. Culture the bacteria and isolate the gene
product (e.g. insulin)
65. Step 1. How to Isolate the Gene of Interest
Use Reverse Transcriptase to make the gene of Interest
Method #1 (see figure on next slide)
1. Isolate mRNA for the gene product of interest (e.g.
Insulin mRNA)
2. Use Reverse Transcriptase to produce cDNA
(complementary DNA)
3. Use PCR to clone the cDNA
3. Separate the synthetic gene of interest by
electrophoresis
66. Use of Reverse Transcriptase
to make complementary DNA
(cDNA) of a eukaryotic gene
67. Types pf restriction enzymes
• Type I and Type II restriction enzymes have both
endonuclease and methylase activity on the same
polypeptide.
• Type I enzymes cleave the DNA at a location of at least
1000 bp away from the recognition sequence.
• Type II . These enzymes cleave DNAs at specific sites
within the recognition sequence.
• Type III enzymes cleave DNA from 24 to 26 bp away
from the recognition site.
69. Cleavage Sites
The red arrows indicate the cleavage sites. The red diamonds indicate the axes of two-fold rotational
symmetry. The blue and yellow colors represent DNA on each side of the cleavage site.
![PCR cont’d
• A convenient shorthand way
• of representing a complete set of reaction conditions
is:
• 94o
C5:00
[94 o
C0:30
; 55 o
C0:45
; 72 o
C2:00
]35; 72 o
C7:00
• which means an initial denaturing step of five minutes
at 94oC followed by 35 cycles of denaturation 94oC for
30 seconds, annealing 55 oC for 30 seconds and
extension 72oC for two minutes and then a final
extension at 72oC for seven minutes.](https://image.slidesharecdn.com/mbb302-microbialgenetics-part22020-250702101908-fc89824e/85/MBB-302-MICROBIAL-GENETICS-PART2-2020-ppt-61-320.jpg)
