1
Q
Bacterial Genome
A
- Usually 1 circular chromosome
- 4-5 Mb of DNA
- Tightly packed, DNA molecule condenses by supercoiling and looping
- Each bacterium replicates and then divides by binary fission into 2 daughter cells.
- Prokaryotes are haploid
- Often contain plasmids
- Only circular DNA molecules in prokaryotes replicate
2
Q
Eukaryote genomes
A
- Diploid
- Linear chromosome, usually more than one
3
Q
Plasmid
A
- Small circles of double stranded DNA
- Used as cloning vectors
- May contain genes that benefit host bacterium or contribute to bacterial pathogenicity
4
Q
Resistance plasmids
A
- Contain transposons
- Carry genes tat confer resistances to multiple antibiotics
- Can be transferred from one bacterium to another in nature
5
Q
Prokaryote genomes
A
- More than 80% of the chromosomal fraction is dedicated to protein coding genes required for growth and metabolic functions
- 1% encodes RNA specifying genes
- The rest comprises intergenic spacers containing regulatory signals
6
Q
Microbial variation
A
- In comparison to eukaryotes, genome sizes in singled celled organisms varies very little
- But bacterial display big variation in metabolic properties, cellular structures and lifestyles
- Phenotypic diversity among species is remarkable
- However organisational features of the genome are very well conserved
7
Q
Organisation of microbial genome
A
- Many cellular processes within bacterial are coupled
- Genes within bacteria are usually co transcribed
- Genes are arranged in OPERONS
- Functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter
- The genes are transcribed together in an mRNA strand
- Genes contained in the operon are either expressed together of not at all
8
Q
Gene transfer in Bacteria:
A
Vertical gene transfer
- occurs in sexually reproducing organisms
- traits are transferred from parent to offspring
Horizontal gene transfer
-Traits are introduced from unrelated individuals or from different species
9
Q
Three mechanism for gene transfer in bacteria. In all three mechanisms..
A
- Donor bacterium provides the DNA that is transferred
- Recipient bacterium receives the DNA, which results in altered phenotype
10
Q
Transformation
A
- Lysis of donor cell releases DNA into medium
- Donor DNA is taken up by the recipient
- Process by which a DNA molecule is taken up from the external environment and incorporated into the genome of the recipient cell.
11
Q
Conjugation
A
- Donor DNA is transferred directly to recipient through a connecting tube.
- Contact and transfer are promoted by a specialised plasmid in the donor cell.
- Temporary direct contact between two bacterial cells leading to an exchange of genetic material (DNA).
- This exchange is unidirectional, i.e. one bacterial cell is the donor of DNA and the other is the recipient.
- In this way, genes are transferred laterally amongst existing bacterial as opposed to vertical gene transfer in which genes are passed on to offspring.
12
Q
Transduction
A
- Bacteriophage infects a cell
- Lysis of donor cell. Donor DNA is packaged in released bacteriophage
- Donor DNA is transferred when phage particles infects recipient cell.
- Involves transfer genetic material from one bacterium to another by a bacteriophage.
- Acting as a vector, the virus carries its own genome plus a fragment of DNA from the bacterium it has recently infected. If the host bacterium survives the viral attack, recombination may occur,
13
Q
Personalised Medicine
A
- Individual respond differently to drugs and sometimes the effects are unpredictable
- Differences in DNA that alter the expression or function of proteins that are targeted by drugs can contribute significantly to variation in the responses of individual
- Genome based information and technologies may yield a new set of molecular diagnostic tools that can be used to individualise and optimise drug therapy
14
Q
The goal of Personalised Medicine
A
- The right dose…
- The right drug for….
- The right indication for….
- The right patient for at…
- The right time…
15
Q
Walfarin
A
- Multisource anticoagulant (different strength tablets)
- Walfarin inhibits vitamin K reductase, which is the enzyme responsible for recycling oxidated vitamin K back into the system. For this reason, drugs in this class are also called vitamin K antagonists.
16
Q
Walfarin doses
A
Narrow therapeutic Index
-small separation between dose response curves for prevent ing emboli and excess coagulation
Non linear dose response (INR)
-small changes in dose may cause changes in INR with a time lag
Wide ranges of doses to achieve target INR of 2-3
17
Q
Walfarin Metabolism
A
- 2 polymorphic genes, CYP2C9 and VKORC1 affect walfarin metabolism and response
- Allelic frequencies of these two genes are usually associated with ethnicity
Concerns with prescribing patients with the 2 polymorphisms
- overdose can result in bleeding which can be fatal
- underdose can thrombosis which can be fatal
CYP2P9 is involved in walfarin metabolism
VKORC1 influences walfarin anticoagulation effect through vitamin K
18
Q
Influenze Subgroups
A
Influenza A
- highly infective
- infects many species
- causes widespread epidemics (pandemic)
Influenza B
- found in humans
- capable of producing severe disease
- causes regional epidemics
Influenza C
- causes mild disease
- humans are natural host, but also found in pigs
- does not cause epidemics
19
Q
Reasons for pandemics
A
- New flu virus occurs due to a process involving mutation and recombination of viral genomes
- Mutations can occur in the replication process of the viral genome
- Mixing occurs because different strains of influenza virus can exchange genes by infecting different animals
- Avian influenza viruses can exchange genes with human influenza viruses creating hybrid stains
20
Q
Influenza Virus genome
A
- RNA virus
- The genome consist of 7-8 RNA fragments, each coding for a viral protein
- 8 genes are responsible for the translation of 10-11 viral proteins.
PB2-transcriptase: cap binding
HA: Haemagglutinin
Na: Neuraminidase: release of virus
21
Q
Influenza virus structure
A
- Nucleocapsid: RNA enclosed in a protein coat
- Surrounded by a lipid envelope
- Two glycoproteins present: HA and NA
22
Q
Influenza Virus Lifecycle
A
- HA binds to cell GP at a Sialic Acid binding site
- Clathrin-coated pit endocytoses virion
- Conformational change: hydrophobic binding of HA to vesicle membrane - RNAs are released into cytoplasm for replication and transcription (vRNA and mRNA)
23
Q
Neuraminidase
A
- Allows release of the newly formed viruses within the host
- Determinant of disease severity
24
Q
Release of Newly Formed Viruses
A
- Replicated vRNA, RNA replicases and other viral proteins are assembled into virions
- HA and NA cluster into a cell membrane bulge
- Virion leaves the nucleus and enters the membrane protrusion
- Mature virus buds from cell in host membrane containing HA and NA
- HA binding virus to cell surface via receptors containing sialic acid
- NA cleaves receptors allowing release of virus
25
Mutation of vRNA
- Absence of RNA proofreading enzymes
- RNA polymerase that copies the genome makes an error every 10 000 nucleotides
- Majority of newly manufactured viruses are mutants
- This enables the virus to surface antigens slowly over time
Leads to ANTIGENIC DRIFT
26
Antigenic Shift
- More than one virus can infect a host cell at one time
- Allows mixing of the 8 separate segments of the vRNA and reassortment
- Rapid change in viral genetics and antigen expression
Leads to ANTIGENIC SHIFT
Both shift and drift allow the virus to evade host immune system
27
Out of African Theory
Suggest that Homo erectus evolved into Homo sapiens in Africa and then ventured out of Africa and dispersed to all around the world.
28
Multi Regional Evolution Theory
Suggests that Homo erectus ventured out of Africa and then evolved into modern man in several different locations through out the world
29
Evidence for Molecular evolution
- DNA sequencing evidence shows that modern humans originated in Africa and migrated north out of Africa then eventually to the rest of the world
- Oldest fossils of modern humans are found in African dating around 16000 years old
30
Genetic Tools
Fossil records
DNA sequencing
- mtDNA: maternally inherited, female side
- Y c/some:paternal line, complementing the mtDNA
- Microsatellite DNA analysis: segments of tandemly repeated DNA with a short repeat length, usually 2-5 nucleotides.
31
MtDNA
-Suggested that modern humans first appeared in Eastern Africa about 150 000 years ago and left between 35 000-89 000 years ago, eventually conquering the globe/
32
Modern Humans using mtDNA
- Based on mtDNA alone, "out of africa" hypothesis was correct with no introgression with local species of Homo
- Most genetic diversity is in Africa
- Ancestral allele are in Africa
- Non africans are nested within the African clade (Non african alleles are a subset of African alleles)
- Supports the scenario that H.sapies originated in African and a small subset migrated out of africa
33
Y chromosome DNA study
- Looked at 1200 DNA samples in Asia
- Looked for 3 specific mutations on Y c/come known to have originated in Africa
- Everyonne carried one of the three mutations or polymorphisms
- Showed little/no interbreeding from Homo erectus and H.sapiens
- Individuals are descendants from Africa
- Likely that the early African humans emigrated to North African and made the lead to Asia and then to the rest of the world
- Indicates that modern humans of African origin completely replaced earlier popn in East Africa
34
Bioinformatics
- Europeans and Asians share 1% to 4% of their nuclear DNA with Neanderthals, but Africans do not.
- Data suggests that early modern humans interbreed with Neanderthals after modern humans left Africa, but before they spread into Asian and Europe.
35
Molecular clocks
- Evolutionary time can be measured with a molecular clock
- Provides a good estimate of time of divergence because rate of evolution is constant across all lineages
- The molecular clock hypothesis states that DNA and protein sequences evolve at a rate that is relatively constant over time and among different organisms
36
Comparing proteins
Two highly conserved proteins are:
Cytochrome C
- Extracts energy from nutrients in mitochondria
- 20/104 aa occupy identical positions in all eukaryotes
Homeobox
- Encodes TFL turn on genes during development to ensure correct anatomical position
- Highly conserved 60 aa
- Mutations: humans, disrupts development of fingers and toes, Cattle, double muscling
37
Human Mitochondria
0dsDNA circular genome
- Maternal inheritance
- Lack of recombination
- Mutates 10x faster than nuclear DNA.
38
Benign Tumours
Do not spread from their site of origin but can crowd out (squash) surrounding cells e.g. brain tumour, wart
39
Malignant tumours
- Can spread from their original site and cause secondary tumours.
- This is called metastasis.
- They interfere with neighbouring cells and can block blood vessels.
40
Why are secondary tumours so bad?
Both types of tumours can rob the body essential nutrients so the tumour can sustain rapid growth and division of the cells
41
Secondary tumours
- To form a secondary tumour, a tumour cell needs to leave the vessel system and invade tissue
- The cell must attach itself to a vessels wall. Once this is done, it can work its way through the vessel and enter the tissue
- Less than 1 in 100000 tumour cells will survive long enough to establish a new tumour,a few survivors can escape and initiate new colonies of the cancer
42
Hallmarks and Characteristics
- Sustaining proliferation signalling
- Deregulating cellular energetics (hallmark)
- Resisting cell death
- Avoiding immune destruction (hallmark)
- Inducing angiogenesis
- Activating invasion and metastasis
- Tumour promoting inflammation (characteristic)
- Enabling replicative immortality
- Genome instability and mutation (characteristic)
- Evading growth suppressor
43
Tumour suppressor genes
- Normal role to prevent cancerous growth eg. RB gene
- Acts as a brake by preventing cells from moving through the cell cycle e.g. RB
Expressed proteins usually have one of the two functions:
- maintain the integrity of the genome by monitoring and/or repressing alteration in the genome -->Checkpoint protein
- Negative regulators or inhibitors of cell division. Their function is necessary to properly halt cell division otherwise cell division is abnormally accelerated.
44
Cell cycle
G1- cell grows in size, prepares for DNA replication
S-DNA replication
G2-Cell prepares for division
M-mitosis
1. Breakdown of nuclear membrane
2. Condensation of chromosomes
3. Attachment of chromosomes to mitotic spindles
45
Cell cycle proteins
- Proteins within the cell control the cell cycle
- Signals affecting critical checkpoints determine whether the cell will divide (cyclins, kinases)
G1, M, G2 checkpoints
46
CDKs and Cyclins
CDKs interact with cyclins and control the cell cycle by phosphorylating other proteins
CDKs=family if kinases that regulate the transition from G1 to S and from G2 to M
- Cyclins specifies the protein target for CDK
- Phosphorylation by CDKs can activate or inactivate a protein.
47
Checkpoint proteins
- Proteins called cyclins and CDK proteins are responsible for advancing a cell through the 4 phases of the cell cycle
- Formation of activated cyclin/cdk complexes can be stopped by checkpoint proteins
- When checkpoint genes are rendered inactive by mutation, the division of normal healthy cells may not be adversely affected.
- In this case, additional mutations e.g. exposure to a mutagen maybe required to affect the function of the mutated checkpoint protein
48
Cell cycle checkpoint ensure genomic stability
Checkpoints monitor the genome and cell cycle machinery before allowing progression to the nest stage of cell cycle
G1 to S checkpoint
-DNA synthesis can be delayed to allow time for repair of DNA that was damaged during G1
G2 to M Checkpoint
-Mitosis can be delayed to allow time for repair of DNA that was damaged during g2
Spindle checkpoint
-Monitors formation of mitotic spindle and engagement of all pairs of sister chromatids
49
Telomeres and telomerases
Telomerase=enzyme (complex of RNA and protein) that adds telomere sequences to the ends of chromosomes
- Loss of control of telomere length may also contribute to cancer
- Normal, specialised cells have telomerase turned off, limits cell divisions
- Cancer cells have to express telomerase to be able to divide indefinitely
50
Genetic basis of Cancer
- Disease characterised by uncontrolled cell division
- Genetic disease at the genetic and cellular level
- Cancer is classified according to what type of cell that has become cancerous
Familial or hereditary-10% cancers: higher predisposition to develop the disease as an inherited trait
Sporadic-most cancers do not involve genetic changes that are passes from parents to offspring.
51
Characteristics of Cancer
- Oiler, less adherent
- Loss of the cell cycle control
- Heritable
- Transplantable
- Dedifferentiated
- Lack contact inhibition
- Induce local blood vessel formation (angiogenesis)
- Invasive
- Increased mutation rates
- Can spread (metastized)
52
Two unifying themes of cancer genetics
Cancer is a disease of genes
- multiple cancer phenotypes arise from mutations in genes that regulate cell growth and division
- Environmental chemicals increase mutation rates and increase chances of cancer
Cancer has a different inheritance pattern than other genetic disorders
- Inherited mutations can predispose to cancer, but the mutation causing cancer occur in somatic cells
- Mutations accumulate in clonal descendents of a single cell
53
Germ line mutations
-Cells that give rise to gametes (egg/sperm)
-Mutations originates in meiosis and affects all cells of an individual
-Increases cancer susceptibility
~50 familial forms
54
Somatic cell
- All cells of the body excluding germ line cells e.g. skin, muscle cells
- Most mutations occur in somatic cells
Mutations: originate in mitosis
- early or during development
- affect limited area of the body
- no transmission to offspring
55
Spontaneous mutations
- Errors in DNA replication
- Toxic metabolic products
- Changes in nucleotide structures
- Transposons
56
Induced mutations (environmental triggers)
- Chemical agents
- Changes in structure of DNA
- Physical agents: damage DNA e.g. xrays, UV light
57
Mutations in proto oncogenes convert them to oncogenes
Four common mutations
1. Missense mutation e.g. ras gene
2. Gene amplification e.g. increase copy number (e.g. myc in lung cancer)
3. Chromosomal translocation e.g. Burkitts lymphoma
4. Viral insertion e.g. cervical cancer
---> GAIN OF FUNCTION
58
Proto-oncogene
=genes that normally trigger cell division.
=active when cell division necessary e.g. in an embryo or during wound healing. They have roles as secreted growth factors, signal inducing kinases and TFs (e.g RAS, MYC)
59
Oncogene
-A gene which is associated with cancer due to mutations
Onco="swelling, mass or tumour"
-Involved directly or indirectly in controlling the rate of cell growth
60
Tumour surpressor genes
- Act in a recessive way
- Normal gene that encodes a protein that helps prevent cancer
- Maintenance of genome integrity
- Negative regulation of cell division
61
Retinoblastoma (Rb)
- Mutation in Rb gene, chromosome 13q
- Rare, cancerous tumour in cone cell of retina. Abnormal appearance of the pupil
- Almost always present in early childhood (birth to 4 years) and is often bilateral
- Rb protein binds transcription factors so that they cannot activate genes that carry out mitosis
- Normally halts the cell cycle G1
- A negative regulator of the cell cycle through its ability to bind transcription factor E2F and repress transcription of genes required for S phase of the cell cycle
- Study of Rb was the origin of the "two hit hypothesis" of cancer causation
62
Two hit hypothesis
-Two mutations or deletions are required
One in each copy of the RB gene
-For sporadic cases: RB is a result of two somatic mutations
For familial cases (inherited)
- individuals harbour one germline mutant allele for the RB gene in each of their cells
- this is followed by a somatic mutation in the normal allele
63
M-/T-tropic HIV
Two types of HIV strain in virus transmission
Early HIV transmission (virus in M-tropic)
- gp120 is able to bind to CD4 and chemokine receptors, CCR5
- found on macrophages
- occurs in 90% cases
Late phase HIV infections (virus is T-tropic_
- gp120 capable of binding to CD4 and chemokine receptor CXCR4
- found on T-lymphocytes
- phenotypic switch from M-tropic
64
Retrovirus Replication Cycle
1. Fusion of HIV to the host cell surface
2. HIV RNA, reverse transcriptase, integrase and other viral proteins enter the host cell
3. Viral DNA is reverse transcribed
4. Viral DNA is transported across the nucleus and integrates into the host DNA
5. New viral RNA is used as genomic RNA and to make viral proteins
6. New viral RNA and proteins move to the cell surface and new immature HIV forms
7. The virus matures by protease releasing individual HIV proteins
65
HIV Genome: Major Genes
gag
= "group specific antigen". Encodes structural proteins, capsid, matrix, nucleoprotein (RNA binding)
```
pol
=encodes enzyme
-proteases cleaves viral polyprotein
-RT/RNases for reverse transcription
-Intergrase cuts cell DNA to insert proviral DNA
```
env= "envelope"
-encodes for envelope glycoproteins; surface, transmembrane
66
HIV Genome: 5' end region
R-terminal 5' end sequence (becomes 3' end of proviral DNA, signal for poly-A addition to mRNA)
PB-primer binding site of cell tRNA
Leader-recognition sequence for packaging genome RNA donor site for all spliced sub-genomic mRNA
67
HIV Genome: 3'end region
PP-polypurine (AUG) tract, initiation site for viral (+) DNA synthesis
U3-unique 3' sequence (becomes 5' end of proviral DNA), regulatory sequence for mRNA transcription and DNA replication
R-terminal repeat, for reverse transcription
68
Retroviral genome
- Retrovirus contains two copies of the RNA genome held together by multiple regions of base pairing
- The RNA complex also includes two molecules of a specific celluar RNA (tRNA lys) that serves as a primer for the initiation of reverse transcription
- The primer tRNA is partially unwound and H-bonding near the 5' end of each RNA genome in a region called the prier binding site.
69
HIV:
| Activation of viral transcription
- Integrated viral DNA may then lie dormant
- ->Latent stage of HIV infection
- ->Last up to 10 years
- Viral replication is triggered
- ->Host cellular treanscription factors are needed
- NF kappa B (NFkB) is very important
- -> unregulated in activated macrophages and T cells
- Cells most likely to be killed by the HIV are those currently fighting infection
70
HIV:
| Genetic Variability-Mutations
- HIV has a very high genetic variability
- Fast replication cycle: generation of about 10^10 virions everyday
- Hight mutation rate
- Generation of many variants of HIV in a single infected patient in the course of one day.
71
HIV:
| Genetic Variability
- Single cell simultaneously infected by two or more different strains of HIV
- Genome is progeny virions maybe composed of RNA strands from different strains
- This hybrid virion then infects a new cell where it undergoes replication
- vRNT jumps back and fourth between the two different RNA templates
- Newly synthesised retroviral DNA sequence that combines the two parental genomes
72
Opportunistic infections Associated with AIDS
Bacterial
- tuberculosis
- strep pneumonia
Viral
- Herpes
- Influenza
Parasitic
- Pneumocytis carinii
- Cryptosporidium
Fungal
- candida
- Cryptococcus.
73
Lambda phage
- Temperate phage
| - Uses both lytic and lysogenic cycles
74
Bacteriophage Replication:
Lytic cycle
Lytic cycle= results in cell lyses and release of progeny phage
- Phage injects its DNA into bacterial cell
- Phage proteins are expressed and take over protein synthesis and DNA replication machinery of infected cell
- Phage DNA replication occurs
- Phage particles are assembled with phage DNA and protein
- Infected cell burst releasing 100-200 viral particles able to infect other cells
75
Bacteriophage Replication:
Lysogenic cycle
- Does not result in immediate lysing of the host cell
- Viral genome integrates with host DNA and replicates along with it fairly harmlessly (prophage)
- Virus remains dormant until host conditions deteriorate, (maybe due to nutrient depletion)
- Prophages activate and initiate the reproductive cycle resulting in lysis of the host cell
- Lysogenic cycle allows host cell to survive and reproduce, allowing the virus to be reproduced in all cell's offspring
76
Lambda
| Circularisation
- Circularisation via cohesive 'sticky' ends (cos ends)
| - Linear phage lambda chromosome in the bacteriophage particle circularises in host cell after infection
77
Lambda phage Genome
The organisation of the genes with this circular structure reflects the two alternative life cycles of the virus
```
Top genes (cI, cro, CIII, O, P) are transcribed very soon after infection, at the beginning either life cycles
-Pattern if their expression determines which of the two cycles prevails
```
Genes of left (N, CII) of viral genome encode proteins that are responsible for the lysogenic infection
Genes on the right (Q, S, R of viral genome encode proteins responsible for the lytic infection
78
Life cycle of Lambda
1. Virus enters cell
2. PL and PR gets activated
3. PL transcribes to make N protein
3. PR transcribes to make cro protein
4. Termination sites stop transcription but when enough N protein is made, transcription goes past these two stop sites (you can now make cIII and cII, replication proteins (O and P) and Q)
6. There are also termination sites next to Q protein. Q protein will allow transcription past this site.
7. If Cro protein blocks production of cI (goes lytic)
8. If cII and cIII activates transcription to make cI (goes lysogenic)
9. cI blocks PL and PR (stops transcription) by binding to OL and OR.
79
Genetic switch: Cellular proteases
- Activity of the cII protein plays a key role in directing lambda to the lysogenic or lytic cycle
- The cII protein is easily degraded by cellular proteases produced by E. coli
- Whether or not these proteases are produced depends on the environmental conditions
80
Cellular proteases choosing lytic cycle
Growth conditions are very favorable, the intracellular levels of the proteases are high
-The cII protein tends to be degraded
-Therefore, PRE cannot be activated and the lambda repressor is not made
-Instead, the cro protein slowly accumulates to high levels
-The binding of the cro protein to OR prevents transcription of the lambda repressor from PRM
-At the same time, the cro protein allows the lytic cycle to proceed
-Thus, environmental conditions that are favorable for growth promote the lytic cycle
This makes sense because a sufficient supply of nutrients is necessary to synthesize new bacteriophages
81
Cellular proteases choosing lysogenic cycle
-If the nutrients are limiting (starvation conditions), the cellular proteases are relatively inactive
-The cII protein builds up much more quickly than cro
-Therefore, the cII protein will turn on PRE
The lambda repressor is made
-Environmental conditions that are unfavorable for growth promote the lysogenic cycle
This makes sense because there may not be sufficient nutrients for the production of new bacteriophages
82
How do cells leave lysogenic cycle and go to the lytic cycle?
-By stress
UV light
- recA (a cellular protein normally involved in DNA recombination) detects the DNA damage
- It is activated to become a protease
- It cleaves the l repressor and inactivates it
- This allows transcription from PR
- Therefore, the cro protein will accumulate
- Favouring the lytic cycle
- The exposure to UV light may have already damaged the bacterium to the point where further bacterial growth and division are prevented
Advance Genetics
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