Lectures 1-10 Flashcards
What type of organisms are prokaryotes?
Bacteria
Archaea
What type of organisms are eukaryotic?
Fungus
Animal
Plants
What causes sickle cell anemia?
Mutant haemoglobin
What causes the swiss cheese phenotype and what is the result of this?
A mutant phospholipase
Causes brain cells to commit suicide
Adult brain is full of holes
What causes obesity, as tested in mice and why does this occur?
Hormone leptin is missing
Causes loss of a cell signalling pathway
What are sugars and what is their function in cells and organisms?
Polysaccharides
Energy source, making nucleotides and forming polysaccharides
What is the molecular formula for monosaccarides?
(CH2O)n
where n = 3 or more
What cause the differences between alpha and beta glucose?
Differ due to mutation
What form do a- and B- glucose chains interchange by?
Linear form
What is glycogen?
Multibranched a-glucose
Polysaccharide
Energy storage in animals, fungi, bacteria (universal)
What is cellulose?
B-glucose molecules linked to form fibres
Give structured cell walls in plants
What is B-glucose polymerised into?
Long linear chain
What is the function of fatty acids in the cell and organism?
Energy - fatty acids
Structure - membranes
Action (catalysis, communication)
What is a lipid?
Water insoluble molecule that is soluble in organic solvents
What is the structure of a fatty acid?
Carboxylic acid group one end
Methyl group the other end
Long aliphatic chain (saturated or unsaturated)
What is the difference in the appearance and structure of a saturated and unsaturated fatty acid?
Saturated: no double carbon bond - straight
Unsaturated: double carbon bond - kinked
What are 2 examples of steroid hormones?
Oestrogen: prepares for ovulation, falling levels triggers ovulation
Progesterone: thickens mucus wall
How many common amino acids are there?
20
What is the standard structure of an amino acid?
Central alpha carbon
Basic amino group (NH2)
Acidic carboxylic group (COOH)
Hydrogen
Side chain - R group
What is the common charge of an amino acid when in the correct pH?
Positively charged
Why is valine insoluble?
The side chain on the amino acid has no solubility
What is another phrase for hydrophilic and hydrophobic?
Hydrophilic: water-loving
Hydrophobic: water-fearing
What is polarity when referring to macromolecules?
A sense of direction
What is the polarity in proteins?
Read from amino to carboxyl
N terminus to C terminus
What are chaperones when associated with proteins?
Making sure other proteins can fold properly, action molecules
What are the functions of nucleotides in cells and organisms?
Action and storing genetic information
What is the polarity in nucleotides?
ready from 5’ to 3’
What are the 3 main domains of life?
Bacteria
Archaea
Eukaryotes
When was there estimated to be a common evolutionary ancestor between all organisms?
3.5-3.8 billion years ago
What is phylogeny?
Relationship between organisms
Visual representation of the evolutionary history of populations, genes or species
What are the 3 types of evidence to construct phylogeny?
Morphology
Molecular evidence
Fossils
What are homoplastic traits?
Traits that are similar for reasons other than inheritance from a common ancestor
What are synapomorphies?
Derived form of a trait shared by a group of related species
Shared derived characteristics
How is molecular evidence used to construct a phylogeny tree?
Take DNA of modern animals and compare to others to see how similar it is to another animal
Who classified the diversity of life and when did they do it?
Linnaeus
1700s
What are the tips in a phylogenetic tree?
The terminal ends of an evolutionary tree
What are nodes?
Points in a phylogeny where a lineage splits
Branching point
Represents where populations became genetically isolated
What are clades?
Single branches - an organism and all its decedents
All organisms that share a common ancestor
Set of hierarchically nested groups
What is morphology and how is it shown in a phylogenetic tree?
Study of size, shape and structure of organisms
Each own characteristic shows a separate trait
What are characters in the study of evolution?
Heritable aspects that can be compared
What are taxa in the study of evolution?
Group of organisms that form a cohesive taxonomic unit
What is homology?
Similarity of traits in different species resulting from their inheritance from a common ancestor
What is the first principle of phylogenetic inference?
Assumes similar features are…
homologous until shown otherwise
What is the second principle of phylogenetic inference?
Doesn’t use shared ancestral features but..
uses shared derived features
What are autapomorphies?
Unique morphological features
Do not provide useful grouping information
Why might microorganisms share genes?
Due to horizontal gene transfer
Where genetic material other than other than from parents to offspring is transferred to another organism
When was Darwin’s theory of evolution?
1859
What can phylogeny be used for?
Used to identify source of viruses
Date of disease onset
Track viral evolution
Identify modes of potential transmission
Organize knowledge of diversity
How do you know if organisms are more closely related in a phylogenetic tree?
Taxa that diverged from each other more recently
Have more character states in common
How can timing be estimated within phylogeny?
Combing phylogenetic morphological evidence with fossils
What are branches?
Lineages evolving connecting other branching events
What are internal nodes?
Occur within phylogeny, represent ancestral populations or species
What is a cladogram?
When phylogenetic tree shows only relationship among species
What does monophyletic mean in phylogeny?
Group of organisms that form a clade
What does polyphyletic mean in phylogeny?
Taxon that doesn’t include common ancestor of all members of the taxon
What does paraphyletic mean in phylogeny?
Group of organisms that share a common ancestor although the group doesn’t include all the decedent of that common ancestor
What is an outgroup?
Group of organisms outside of monophyletic group
What is homoplasy?
Character state similar not due to shared descent
What is convergent evolution?
Independent origin of similar traits in separate evolutionary lineages
What is evolutionary reversal?
Describes reversion of a derived character state to form resembling its ancestral state
What is parsimony?
Selection of alternative hypothesis
Require fewest assumptions or steps
What is polytomy?
Internal node of phylogeny with more than 2 branches
What is a consensus tree?
Combining hypotheses
What is exaptation?
Trait that initially carries out one function and its later co-opted for a new function, original function may be lost
What does LUCA stand for?
Last universal common ancestor
What are the 3 major divisions or domains of life?
Bacteria
Archaea
Eukaryotes
When was the last LUCA?
3.5-3.8 billion years ago
How are the 3 major divisions or domains of life identified?
Based on comparison of ribosomal RNA
Who established the 3 domains of life and when?
Carl Woese and George Fox
1977
Who revised the ‘web of life’ and when?
W Ford Doolittle
2000
How did W. Doolittle revise the ‘web of life’?
Domains of life are based on horizontal gene transfer
Through the sharing of genes
A community rather than a single origin
In what ways are eukaryotes similar to archaea?
- Proteins involved in cytokinesis
- Cell shape determination
- Protein recycling
- Membrane remodelling (cellular compartmentalisation)
What was previously understood about similarities between archaea and eukaryotes?
Features used to be unique to eukaryotes ONLY
What is the most modern view of the ‘tree of life’?
Hug et al
2016
Based upon the sequences of 16 ribosomal proteins, extensive mapping
Eukaryotes are group that has arisen from archaea
When was Einstein’s Theory of General relativity?
1915
What is the general relativity theory?
Gives the simplest description of cosmology that is consistent with all known experimental and observational data
Consistent across all known data
Why is Einstein’s work under scrutinity?
Echoes of gravitational waves when black holes fuse
Suggests there is a structure to the event horizon of a black hole
(Einstein suggest nothing should be found there)
What is the movement of information to understanding science starting with Mythos and then Logos?
Mythos: stories of gods and heros
Logos: dominated by rational thinking
- lack of evidence for claims
- Science is performed in a naturalistic framework
What are the fundamental principles of science?
- Science is performed in a naturalistic framework
- Experimental/ observational support is required
- Proceed with the simplest explanation consistent with all the data
- Inductive reasoning allows you to draw conclusions
- Compile and disseminate your knowledge
- Accept uncertainty
Who stated that a hypothesis must be supported by experiments based on evidence?
Alhazan
c965-c1040
What is Occam’s razor?
Take the simplest possible view that aligns with data
Supported by Einstein - make simple but no simpler
What is the principle of Parsimony?
Theories should explain and predict with the fewest assumptions
What percentage of papers are rejected?
80%
What must happen before work is published?
Scientists describe their approaches, observation and conclusions in sufficient detail allowing others to extend or repeat them
What must happen before work is published?
- Scientists describe their approaches, observation and conclusions in sufficient detail allowing others to extend or repeat them
- Papers are submitted to journal
- Papers returned for improvement
What is the idea of falsification with science?
Science is a set of ideas about how the universe works
Science has an in-built self correcting mechanisms
We can never be certain that new data will not change out ideas - all science is theory
What is evolution?
The genetic content of a population changes over time
What is microevolution?
Changes in allele frequencies in a population of a species over time
What are the 3 main mechanisms of microevolution?
Natural selection
Genetic drift
Gene flow
What is macroevolution?
Changes at or above the level of the species
What was the second voyage of HMS Beagle?
5 year journey but 3 years on land
Darwin - Galapagos Islands
What ideas influenced Darwin?
Economic population (population growth and food supply)
Naturalistic framework
Observations and prior knowledge
Combination of all factors contributes to natural selection
Who tested Darwin’s postulates hypothesis?
Rosemary and Peter Grant
1976-1978
How did Rosemary and Peter Grant test Darwin’s first
and second postulate?
Off coast of Ecuador in the Galapagos islands
Finches show variation in beak length so all captured and measured
Variation to support: parents with small beaks have offspring with shallow beaks, parents with deep beaks tend to have offspring with deep beaks
Large genetic component - heritable component with beak length
How does Rosemary and Peter Grant’s experiment support Darwin’s third postulate about reproduction not being random?
Individuals that reproduce the most are those with the most favourable variations
Beak depth increased, deeper beaks can cracker harder seeds which is advantageous
How does Rosemary and Peter Grant’s experiment support Darwin’s second postulate about some variation being heritable?
Variation to support
Parents with small beaks have offspring with shallow beaks, parents with deep beaks tend to have offspring with deep beaks
Large genetic component to determine beak length
How does Rosemary and Peter Grant’s support Darwin’s first postulate about variety within species?
Medium ground finches showed variation in beak depth
How has genetic variation been shown in the Chernobyl nuclear disaster with frogs?
Melanin protects against ionising radiation
Brighter frogs in background radiation areas
Darker frogs in high radiation conditions
What is the conclusion about Darwin hypothesis and microevolution?
Populations adapt genetically to the environment
Microevolution by natural selection is a theory
What is genetic drift?
Changes in allele frequencies in a population
What percentage of genes are protein coding genes?
1.5%
Why are mutations not always acted upon?
As many are not in coding genes
(only 1.5% of protein coding genes in the genome)
Mutations from natural selection not acted upon
What are overall trends in natural selection and allele fixation?
Dominant: rapid rise as seen in both heterozygote and homozygote
Recessive: slower rise - only visible in homozygotes so fixation takes longer
How can genetic drift occur by sampling error?
Not every sperm contributes to next generation
Massive attrition occurs due to death
Sampling error is causing evolution to happen rapidly
How did Richard Lenski measure long term evolution?
E. Coli grown in medium with glucose and citric acid for carbon source
Sub-cultured every day for 30 years, every 75 days samples taken and frozen as fossil records
3 flasks developed mutations and some cells affected DNA repair mechanisms causing mutant streams
How did citrate used in aerobic conditions evolve in Lenski long term experiment?
Citric receptor only expressed in anaerobic conditions
In aerobic conditions transporter is not expressed so E.Coli cannot utilise external citrate as an energy source
Gene is duplicated by mutation causing strains being able to utilise citrate as well as glucose as energy sources
What is gene flow?
The movement of alleles between previously separate populations
How can alleles move by gene flow?
Migration of adults and subsequent mating
Movement of gametes and subsequent fertilisation
Alleles migrating from one area to another and finding mating success
What are the 3 main mechanisms to gene flow?
- Genetic drift removes genetic variation within demes - causes differentiation between demes
- Gene flow introduces new alleles into demes within a metapopulation - can cause genetic homogeneity between demes
- Selection and reproductive isolation
What is a deme?
A sub-population
What is a meta-population?
Overall population
What is a species (at eukaryotic level)?
A population of organisms that can potentially or actually interbreed, giving viable fertile offspring
How is each species defined?
Defined on looks and how they act
Not breeding behaviour
What is the argument about tree frogs and chromosomal duplication with species?
Instant speciation occurred when a treefrog failed to sort its 24 chromosomes during meiosis, generating a new species
Identical in size, shape and colour to original but has 48 chromosomes and a different mating call
Gene exchange has occurred
What is the example of the tube mosquito with species differentiation?
Mosquitos adapted to having mice as host
Originally eggs lay in open spaces, multiple genetic changes to host on mammals
Londoners hid in tubes during blitz and caused the plague
How is the ‘Big Bird Lineage’ an example of macroevolution?
Immigrant bird found female partner
2 of the hybrid offspring mated together to produce fertile offspring
Who produced the first complete genome sequence of any organism?
J. Craig Venter
How many base pairs were sequenced by J. Craig Venter?
1.8 million
When was the full human genome sequenced?
2003
When was all human genome set to be sequenced by?
Between 2008-2015
1,000 genomes project - describe all human genetic variation
When could gene mapping be done with detecting the likelihood to diseases?
2012-2018
On what date was the first draft of the complete genome sequence reported?
14th April 2003
What are the goals of the human genome project?
- Identify all genes
- Determine sequences
- Store information
- Improve tools for analysis
- Transfer related technologies
- Address ethical, legal, social issues
When identifying genes how many were expected and how many were found?
100,000 genes expected
20,000 found found
Only 1.5% of our genome is protein coding
How big is the haploid human genome?
3,2000,000,000 bp
(3.2 x 10^9)
What is a chromosome fusion event?
2 chimp chromosomes fused in man
What are the benefits to human genome project?
Understanding our evolutionary history
(Human and chimpanzee genomes can be compared to identify genes that contribute to uniquely human traits)
Personalised medicine
(AI that plans how to treat people based on their genome)
To identify functions of all our genes
(Medicine can become predictive, preventative, personalised)
When was there a genomic test for breast cancer risk?
2019
What are the ethical or legal issues associated with the human genome project?
Could be refused employment
Could be denied employment
What is the central dogma of molecular biology?
Describes the transfer of information from DNA via an RNA intermediate to protein
Information cannot be transferred back from protein or nucleic acid
Recognised DNA replication and RNA replication also transfer information
Who published The Central Dogma and when?
Francis Crick
1958
What are the 5 steps to the Central Dogma of molecular biology?
- DNA replication
- Transcription
- Translation
- RNA replication
- Reverse transcription
Who was Gregor Mendel and what was he known for?
Demonstrated the inheritance of certain traits follow particular patterns
Referred to the laws of Mendelian inheritance
The Father of Genetics - naturalistic framework
Why did Mendel work with peas?
They produce large numbers of offspring
They have a relatively short generation time
Both self-fertilisation and cross-fertilisation are possible
Pure-breeding lines with contrasting features were available
What was Mendel’s experiment with peas?
Monohybrid cross - plants with green peas crossed with those with yellow
Only yellow seeds produced (called a dominant trait)
Yellow offspring (Heterozygous) mixed with pure green
Peas were mixture of green and yellow (1:1 ratio)
Called a recessive trait
What was the explanation to Mendel’s first law of inheritance?
Each individual has 2 factors for each trait, one from each parent
If the 2 factors are identical, the individual is homozygous for the trait
If the 2 factors have different information, the individual is heterozygous
Alternative forms of a factor are called allelomorphs (alleles)
What is the Law of Segregation?
2 coexisting alleles of an individual for each trait segregate during gamete formation so that each gamete gets only one of the 2 alleles
Gametes fuse randomly so there is a discrete inheritance of a trait than than a blending
When was the chromosome theory of inheritance and who by?
1902
Walter Sutton
What was the chromosomal theory of inheritance?
Sex was determined via chromosome-based inheritance
Proposed Mendel’s factors were carried on chromosomes
How was sex-linkage detected?
1910, Morgan
Wild-type fruit flies have red eyes but some of his mutant flies has white eyes
Red-eyed females were crossed with white-eyed males = gave all red-eyed offspring
White-eyed females crossed with red-eyed males gave red-eyed females and white-eyed males
Results were influenced by sex of parents - concluded genes for eye colour was carried on the X chromosome
What was Garrod’s ‘inborn errors of metabolism’?
Each case an inheritable factor for a metabolic step was defective
Albinism: lack of pigment
Alkaptonuria: individuals secrete homogentisic acid into their urine, which goes black following exposure to air
Why did Beadle and Tatum use red bread mould in their experiment?
Grows rapidly on a very simple medium containing only salts, C and N
What was Beadle and Tatum ‘one gene-one enzyme’ experiment?
Hereditary diseases are ‘inborn errors of metabolism’ is correct
- Mutagenesis: took haploid and illuminate it
- Grow all survivors in complete medium
- Identify mutants: every survivor transferred to minimal medium, failure to grow identified a potential nutritional requirement
- Identify nutritional requirement: growth on minimal medium containing amino acids identifies a requirement for an amino acid
- Identify arginine auxotrophs
What is a auxotroph?
A mutant that requires a particular additional nutrient
What is a prototroph?
The normal strain which does not require that nutritional supplement
What was Beadle and Tatum’s key steps to their experiment in 1941?
- Illuminate and breed sample
- Transfer some of complete to minimal medium
- Work out whether it has required amino acids
- Test each one for the specific amino acid
What did Beadle and Tatum identify when testing the arginine auxotrophs?
If the auxotroph came from different asci, they would probably have different mutations
If the auxotroph came from one ascus, they have the same mutation
How does complementation work in Beadle and Tatum’s experiment?
As the heterokaryons both contain nuclei, each cell can perform combine phenotypes - each defect complements the other
Suggests they isolated 3 classes of mutants defective in arginine biosynthesis
What other functions do enzymes perform?
Structural and immunological functions
Why did the one gene-one enzyme hypothesis change and what did it change to?
One gene-one protein
Some proteins are not enzymes but perform other functions (structural, immunological)
Multiple genes for a pathway
What did Friedrick Miescher discover in 1869?
Found the ‘nuclein’ which contianed C, N and H prtiens but was rich in phosphorus with no detectable sulphur
Proposed that the nuclein might be the basis of heredity
Who discovered transformation in 1928?
Frederick Griffith
Showed bacterial transformation (bacteria changes its form and function through the action of a transforming principle or transforming factor)
What is the Griffith experiment (1928) - first control?
- Mouse injected with living R cells
- Mouse remains healthy
- Living R cells can be recovered from mouse heart tissue
= R cells survive in mice, but do not cause pneumonia, R cells are non-pathogenic
What is the Griffith experiment (1928) - second control?
- Mouse injected with living S cells
- Mouse contracts pneumonia
- Living S cells can be recovered from mouse heart tissue
= S cells survive in mice, and cause pneumonia, S cells are pathogenic
What is the Griffith experiment (1928) - third control?
- Mouse injected with heat-killed S cells
- Mouse remains healthy
- No living streptococci can be recovered from mouse heart tissue
Only living cells are causing disease
= Dead S cells do not cause pneumonia
In the Griffith experiment, what is the experimental arm?
- Mouse injected with mixture of heat-killed S cells and living R cells
- Mouse dies
- Both R and S cells can be recovered from mouse heart tissue
= An S cell transforming principle that survives heat treatment has altered some of the R cells
Who did work finding the transforming principle and when?
Avery, MacLeod and McCarthy
1944
What was the Avery, MacLeod and McCathy experiments?
Formal evidence that DNA is a transforming factor
- Living S cells in a flask and boiled to kill cells
- Only soluble extract remains, split into 3 and mixed with protease, RNase or DNase enzyme treatment
Protease: transformation of R cells to S cells - protein is not the hereditary material
RNase: S and R cells = still transformation - cannot be RNase
DNase: no DNA - lost during transformation
= DNase destroys the transforming principle
What is another piece of evidence conducted in 1944 with the Avery, MacLeod and McCarthy experiment?
Purifying DNA from S cells resulted in transformation of some R cells to S cells
Definitive evidence that DNA is the hereditary material
Who was A. Hershey and what did he do?
1952: protein is not the hereditary material
1969: gained Nobel prize for discoveries but did not mention Martha Chase (research assistant) in his acceptance speech
What is the structure of a T2 phage?
DNA - hereditary information, tightly packed
Inside icosahedral head, attached to a core
Surrounded by a sheath
Attached to base plate with tail fibres emerging
What is the state of phage research from 1948-1952?
Taking E.Coli and infecting with T2 causes attachment mediated by the fibres
Phage particles remain attached to the bacterium, heads appear empty forming ‘ghosts’
Bacterium burst open exposing new viruses
What did Martha Chase experiment with the state of phage research in 1952? (Hershey-Chase experiments)
Phage protein labelled radioactively
Sulphur: present in proteins, not DNA
Phosphorus: present in large amounts of nucleic acid
Grow bacteria and infect, when burst spin out dead and non-infected cells leaving a suspension of radioactive phage
Next generation are radioactive - confirms DNA is genetic material, only when labelled with phosphorus
Who was Phoebus Levene and what theory did he propose? (1909)
Tetranucleotide theory
Showed each building block of DNA is a nucleotide:
Phosphate group linked to a deoxyribose sugar, linked to nitrogenous base
What sugar is used in DNA and RNA?
DNA: pentose (5C) deoxyribose
RNA: pentose (5C) ribose
What is the difference in structure between ribose and deoxyribose molecular structure?
Hydroxyl group in ribose on 2’
Hydrogen in deoxyribose on 2’
What bases are purines?
Adenine
Guanine
What bases are pyrimidine?
Cytosine
Thymine
Uracil
What are nucleosides and where/what is the bond?
Sugar and base
Glyosidic bond: between C-1’ and N-9 (purine) or N-1 (pyrimidine)
What are nucleotides and where/what is the bond?
Phosphorylated nucleosides
Ester links: between sugar C-5’ group and the phosphate
What forms do nucleotides come in?
All come in mono, di and tri phosphate form
What kind of molecule is DNA?
Polynucleotide with polarity
What is the polarity associated with DNA?
Phosphodiester bond links the 3’C of one nucleotide to 5’ of the next
Base sequence is written and read 5’ to 3’
Polarity at the 5’ phosphate end –> 3’ hydroxyl end
How did Levene ‘tetranucletoide model’ not support Avery et al?
Showed how DNA was simple and repetitive and could not be the genetic material
Not believed to be transforming principle
Who was Erwin Chargaff and what did he propose?
Nucleotides in DNA should be present in equal proportions
(If Levene was correct)
%T = %A = %G = %C
How did Chargaff show Levene to be incorrect?
Measured concentrations of each of 4 nitrogenous bases in different organisms
Different organisms had different DNA constitutions
Reality: %T = %A and %G = %C
What did Linus Pauling say about DNA? (1951)
Described the alpha helix
Basic structure present in many proteins
Used X-ray crystallography - so shape must be helical
How does an X-ray crystallography indicate the helical structure of DNA?
Crystalline target molecule diffracts X-rays
Causes exposed patches on photographic film
Resulting diffraction pattern is unique
What did Huygens show with the ‘Wave theory of light’?
When light passes through a small opening, a wave front is propagated on the other side, single spot appears on a screen
Wider slit, all points across the slit act as point source - results in single slit diffraction pattern on screen
2 slits causes interference - causes double slit interference pattern on screen
With a diffraction grating the pattern becomes much sharper
What did Augustin Fresnel extend the ‘Wave theory of Light’ concept?
Showed diffraction occurred around a solid object with the same width as a slit
Replacing slit with object of same size causes the same results
What are the spatial relationships with the light theory?
Features that are close produce widely separated reflections
Features that are distant produce closely separated reflections
Vertical grids = horizontal spots
Grid = cross
What did Maurice Wilkins do?
Stretched DNA and air dried it
Allowing for stretching into long fibres and mounting in front of X-ray source
What was Maurice Wilkins produce?
1950 - Produced X-ray diffraction of dried DNA
High resolution
Without clear model in mind - too difficult to interpret
What was Gosling and Franklin B model of DNA?
1952 - Used hydrated DNA after 100 h of exposure
Cross is unmistakable - DNA must be helical
Who proposed the triple helical model of DNA?
Linus Pauling
Triple helix 3-strand model, with bases pointing outwards
(supported by Watson and Crick)
What did Watson, Crick and Wilkins propose?
Double helix 2-strand model, with the bases pointing inwards
What was each turn of the DNA helix measured to be?
3.4nm
(Spots far apart give features that are close together)
How many bases were there thought to be per turn of the DNA helix?
10 bases per turn
(More distant the spots, smaller the actual distance in the target)
What was the measured diameter of DNA to be?
2nm
Calculated using degrees of rise within the ‘X’
Why did Pauling model of DNA fail?
(triple helix)
The negative charges of the stacked phosphate groups would repel each other and destabilise the molecule
- Sugar phosphate backbone must be compressed together
How did Watson’s deduction support Chargaff rule?
2 purines = too wide for DNA
2 pyrimidines = too narrow for DNA
= A must pair with T
= G must pair with C
What was Watson and Crick’s model in 1953?
Made DNA with metal scraps, almost 2m tall
Watson: specific A/T and G/C pairing scheme
Crick: idea of antiparallel strands
Everything clicked into place beautifully
What are 6 key features of Watson-Crick model?
- Right-handed (clockwise) double helix
- The strands are anti-parallel 5’ –> 3’ and 3’–> 5’
- Sugar-phosphate backbones are on the outside of the helix, bases oriented towards the central axis
- Complementary base-pairing - bound by weak hydrogen bonds
- Base pair distance (10.5 bp per turn, helix turn = 3.6nm
- major and minor grooves - backbone not equalling spaced causing grooves
How many bonds does A-T have?
2 hydrogen bonds
What are the other structural variants of DNA?
A-DNA: occurs in low hydration conditions (uncertain whether this occurs)
B-DNA: most structurally stable
Z-DNA: taken up physiologically by stretches of alternating pyrimidines and purines
Who established the directionality of DNA synthesis?
The Okazaki
What is the leading and lagging strand?
Leading: synthesised continuously - same direction as the replication fork
Lagging: synthesised discontinuously - 5’to4’ synthesis proceeds in the opposite direction
What are Okazaki fragments?
Small fragments of strand on the lagging strand only
Consequence of synthesis of new DNA in one direction only
Occur away from replication fork
Why is there no 3’to5’ synthesis of DNA?
One nucleotide removed leaving 5’ phosphate but a triphosphate is required
No high energy bond can be cleaved, no reaction can be processed
High energy bond required for incorporation of nucleotide
What is the life cycle of a M13 bacteriaphage?
Injection via the pilus
Single stranded DNA is replicated and becomes double stranded
Packed into fresh phage and secreted
What did Okazaki and Kornberg discovery with the beginning to DNA replication?
DNase cannot completely destroy Okazaki fragments
Primer for an Okazaki fragment is RNA, not DNA
Little pieces of RNA, 10-12 bases long were left
How are RNA primers synthesised?
DNA primase is a rifampicin-sensitive DNA directed RNA polymerase
Synthesizes an RNA primer to initiate DNA synthesis on lagging strand
RNA polymerases don’t require a primer
How is the lagging strand synthesised?
- New RNA primers are synthesised by DNA primase
- DNA Pol III extends RNA primer using dNTPs to make Okazaki fragments on lagging strand
- As replication fork separates fork separates more DNA, new primers are laid down by DNA primase
- Old primers erased by 5’to3’ exonuclease
- Gap sealed with DNA ligase, joining Okazaki fragment to growing chain
How is the Okazaki fragment joined by DNA ligase?
DNA ligase uses ATP, releasing pyrophosphate and attaching AMP to 5’ phosphate of downstream fragment
AMP released, phosphodiester bond formed between 3’ -OH of upstream Okazaki fragment and 5’ phosphate of downstream fragment
Sealing needs ATP
New bond seal gap
What is the clamp holder?
Hold 2 molecules of Pol III
Has helicase and DNA primase
What is leading strand synthesis?
- DNA helicase unwinds DNA helix, separating strands
- DNA primase synthesises DNA primer on leading strand template
- Primed duplex is captured by Pol III
- New clamp halves maintain in clamp holder
5.Clamp holder transfers 2 halves of B-clamp to Pol III
- Helicase continues to unwinds, and Pol III replicates the leading strand continuously
What is processivity?
Measure of an enzyme’s ability to catalyse consecutive reactions without releasing substrate
What is particular of Pol III?
Has low processivity
Can only make short stretches of DNA before it falls off DNA
What is the process of lagging strand synthesis?
- DNA primase produces RNA primer
- Primed duplex is clamped by Pol III forming loop
- Helicase continues to unwinds, Pol III extends new primer on lagging strand until Okazaki fragment has been pulled back to Pol II
- Lagging strand and template are unclamped
- DNA primase primes the lagging strand template
- DNA Pol I and DNA ligase repair the gap
- Process restarts by clamping new lagging strand primer
Why does DNA Pol III have to have low processivity?
If it was a highly processive enzyme, it could not release the new Okazaki fragment easily
Does DNA Pol I have low or high processivity?
Low
When can stem-loop structures be formed?
When DNA is denatured and fails to re-anneal properly
What is SSB and what is its importance?
Single stranded DNA binding protein
DNA replication required a supporting cast of SSB
It protects the ssDNA from base pairing and from nuclease
Being displaced by Pol III and replaced as the helix is unwound
Why is most DNA negatively supercoiled?
Easier to replicate
Topoisomerases are used to regulate the degree and type of supercoiling
Arises from unwinding of DNA
How is overwound positively supercoiled DNA turned into underwound negatively supercoiled DNA using Type II topoisomerase?
- Topo II binds to the positive supercoil
- Binding and then cutting BOTH strands of DNA
- Brings coil forward and re-ligates it causing negative supercoil
How is negatively supercoiled DNA relaxed using Type I topoisomerase?
- Topo I binds to negative supercoil
- Cuts one DNA strand
- Causes DNA to unwinds and re-ligates it causing relaxed DNA
How is bacterial DNA polymerisation bi-directional?
2x Pol II complexes enter DNA at an origin of replication
Proceeds in both directions at the same time
Topo IV separates the catenated daughter chromosomes by a double stranded break and relegation
How quickly do eukaryotic DNA Pols polymerise at?
50 nucleotides per second
Why do chromosomes get shorter with each replication?
When DNA polymerase falls off and Okazaki fragments are left so they can join
Primers are erased and gap is filled by DNA polymerase and repaired by DNA ligase on leading strand
Gap on lagging strand cannot be filled by DNA polymerase as there is no primer
How is telomerase a reverse transcriptase?
Telomerase provides an RNA template to synthesise a DNA copy of the template at the 3’ end of the parental lagging strand template
Telomeres are build of repetitive motifs
Where is telomerase active?
Some germline cells
Epithelial cells
Haematopoietic cells
and in >90% of cancer cell lines
- responsible for immortal phenotype of cancer cells
Who determined the order of nucleotides in pieces of DNA?
Gilbert and Sanger
What does Sanger sequencing rely on?
The incorporation of dideoxynucleotides into newly replicated DNA
What is a dideoxynucleotide?
Terminator
Lacks a 3’ hydroxyl so is a terminator of chain extension
Allows no more nucleotides to be added
What is needed for Sanger’s dideoxy sequencing method?
Single stranded template DNA
Primer complementary to template
DNA polymerase
Pool of normal deoxynucleotides
Small proportion of radioactively-;abelled ddATP
What is the practice of the Sanger sequencing method?
- Add template + primer + DNA Pol I + dNTPs
- Add appropriate ddNTP
- Separate nested fragments on basis of size by electrophoresis
- Autoradiograph and read sequence from bottom upwards
- Looking for competition between normal nucleotides and incorporation of added product
What is the process of automates dideoxy sequencing?
- Template + primer + DNA Pol + all dNTPs + all fluourescent ddNTPs in one tube
- Dye present in each synthesized fragment corresponds to dye attached to dideoxynucleotide that terminated the synthesis of that particular fragment
- Pass nested product through an electrophoretic system and read with lasers
Who discovered the PCR?
Kary Mullis
What did Mullis say were the components of PCR?
Template dsDNA with target area
2 specific oligodeoxynucleotide primers
dNTPs
Buffer and MgCl2
Taq polymerase
Why is Taq polymerase useful?
Thermal stability of Taq DNA polymerase
What is the method of PCR?
- Heat to 94 degrees to denature DNA and cause H bonds to break
- Cool down (45-65 degrees) to allow primers to anneal
- Warm again to 72 degrees to allow primers to be extended
Repeat 30 times
What happens in cycle 1 of PCR?
- Target DNA is denatured
- Reaction is cooled, allowing primers to anneal to their complementary sequences
- Reaction is raised to 72 degrees, allowing Taq polymerase to extend the primers
- Products of cycle 1 can now act as substrates for cycle 2
How does PCR have both specificity and sensitivity?
Specificity provided by primers:
- Complementary to opposite strands with 3’ ends pointing towards each other
Sensitivity: one target molecule can be amplified to >10^9 molecules in just a few hours, product in one acts as template for next
Why do the regions defined by primers increase exponentially?
Starting DNA and hybrid duplexes also acts a templates for more PCR amplification
Expect >10^9 molecules of product from each starting target DNA after 30 cycles
What are the applications of PCR?
Medical-genetic purposes
Analysing pregnancy tissue for pre-natal genetic screening
Archaeological and ancient DNA sources
Use PCR to alter a DNA sequence
Forensic applications
How were the sequencing on archaic hominin genomes useful?
Found Europeans and Asians have 4-5% of their genes derived from Neanderthals
Gene flow can be detected from Neanderthals into modern humans
(later version: Europeans share 2% of DNA with Neanderthals)
What did Green et al study with sequencing of archaic hominin genomes?
Nuclear DNA was amplified from fossil Neanderthal bones from Croatia
Compared DNA from modern humans
What new type of human was found in 2021?
Eurasian human
Dragon Man
What is the application of PCR with mutations?
Mutations can be introduced into amplified DNA by engineering one or more mismatches in a primer close to its 5’ end
What is a VNTR?
Variable number tandem repeat
Short run of repeated nucleotides sequences
Found on many chromosomes, show variation in length between individuals
How can a VNTR be used for personal or parental identification?
Each VNTR can act as an inherited allele
Number of repeats is variable
Individuals inherit a different variant of each VNTR locus from mother and father
Different sized fragments can be separated electrophoretically
What is an example of VNTR analysis in practice?
CODIS: combined DNA index system, operated by FBI
STR: short tandem repeat, particular form of VNTR
Multiplex PCR is performed using specific primers for 13 different VNTRs on different chromosomes
