Lecture 16 DNA Hereditary Flashcards
How did dye that binds to DNA and turned it red provide circumstantial evidence that DNA was the genetic material?
- It was in the right place
- It varied among species
- It was present in the right amounts
In what ways was DNA present in the right amounts in order to be the genetic material?
DNA is somatic cells was twice that of reproductive cells
On what organism was Frederick Griffith studying in the 1920’s?
Streptococcus pneumoniae (causes pneumonia)
What two strains of pneumonia was Griffith working with?
S strain and R strain
What did cells of the S strain of S. pneumoniae produce?
Colonies that looked smooth and was covered by polysaccharide capsule
What is the significance of the polysaccharide capsule covering S. pnuemoniae?
They are protected from attack by the host cells immune system
What happened when S strain was injected into mice?
Caused pneumonia- strain was virulent
What did the cells of the R strain of S. pneumoniae produce?
Colonies that looked rough (lacked capsule, not virulent)
What did Griffith do to some mice?
Inoculate them with heat-killed S. pneumoncocci (heat killed bacteria does not cause infection)
What happened when Griffith inoculated some mice with a mixture of living R bacteria and heat killed S bacteria?
The mice died of pneumonia
What happened when Griffith examined the blood of mice that died after being inoculated with a mixture of living R bacteria and heat killed S bacteria?
Full of living bacteria, many with S strain characteristics
What was Griffith’s conclusion?
A chemical substance from one cell is capable of transforming another cell
At the time, what was the chemical that could cause a heritable change in affected R cells called?
a chemical transforming principle
Who identified the transforming principle?
Oswald Avery and his colleagues
What did Oswald Avery and his colleagues do to identify the transforming principle as DNA?
They treated samples known to contain pnemococcal transforming principle in a variety of ways to destroy different types of molecules (proteins, nucleic acids, carbohydrates, lipids) and tested samples to see if they had retained transforming activity
What did Oswald Avery and his colleagues find in their experiments?
If DNA was destroyed, transforming activity was lost, but there was no loss of activity when other molecules were destroyed
What was the final step taken by Oswald Avery and Colin Macleod and Macyln McCarty in their experiment?
To isolate virtually pure DNA from the sample containing pnuemococcal transfomring principle and showed that it caused bacterial transformation
Why did the work done by Avery, MacLeod and McCarty have little impact when it was published?
Scientists did not believe DNA was chemically complex enough to be genetic material
Bacterial genetics was new- it was not clear bacteria even had genes
What enzymes were used in Avery’s experiments?
RNase
Protease
DNase
What did the Hershey-Chase experiment sought to determine?
Whether DNA or protein was genetic material
What was the Hershey-Chase experiment carried out with?
Bacteriophage T7- consisting of DNA inside a protein coat
What did Hershey and Chase deduce about the entry of some viral components?
Entry of some viral components affects genetic program of the host bacterial cell, transforming it into a bacteriophage factory
How did Hershey and Chase trace the components of the bacteriophage over its life time?
Radioactive isotopes
What radioactive isotope is used to mark protein?
Sulfur 35
Why is sulfur 35 used to mark protein?
Sulfur is in the amino acid cysteine and methionine
How did Hershey and Chase label the bacteriophage with sulfur 35?
They grew the bacteriophages in the presence of S35
What element is rich in DNA and where?
Phosphorus
In the deoxyribose-phosphate backbone
What radioisotope is used to mark DNA?
Phosphorus 32
What method did Hershey and Chase use to test their hypothesis?
32-P labelled bacteriophage infects bacteria in one experiment, 35-S labelled bacteriphage infects bacteria in the other
What happened after the two different experiments were infected with labelled bacteriophages?
After a few minutes, the mixture was agitated in a blender without bursting bacteria to strip away parts of the bacteriophage that had not penetrated the bacteria
What happened after blending the mixture in the Hershey-Chase experiment?
The centrifuged it to separate the bacteria from the rest of the material
What did the scientists find after centrifuging the experiments?
Supernatant contained mostly sulfur (and thus viral protein) and phosphorus had remained mostly with the bacteria in the pellet
What did the distribution of sulfur and phosphorus in the centrifuged supernatant/pellet suggest?
DNA had been transferred to bacteria, thus DNA was the compound responsible for redirecting genetic program of bacterial cell
What other experiments did Hershey and Chase perform?
Longer range experiments in which progeny viruses were collected- almost none contained labelled sulfur, but about 1/3rd had original phosphorus
What was the logical conclusion to Hershey and chase’s longer range experiment?
Because DNA was carried over in the virus from generation to generations, protein was not, the hereditary information of the virus is contained in the DNA
What is genetic transformation of eukaryotic cells by DNA called?
Transfection
How is transfection demonstrated?
Using a marker
What is a marker?
A gene whose presence in the recipient cell confers an observable phenotype
What marker gene did researchers use to demonstrate transfection in eukaryotic cells?
Nutritional or antibiotic resistance marker genes that permit the growth of transformed recipient cells but not nontransformed cells
What is thymidine kinase?
An enzyme needed to make use of thymidine
What happens in the absence of the gene that codes for thymidine kinase?
Mammalian cells do not grow
What happens when DNA containing thymidine kinase marker gene is added to a culture of mammalian cells? What does this show?
Some cells will grow, demonstrating they have been transfected with the gene
What is a whole new genetically transformed organism called?
A transgenic organism
What was crucial evidence for deciphering the structure of DNA?
X-ray crystalography
What X-ray crystalography?
Isolated chemical substances that form crystals, positions of atoms can be inferred from pattern of diffraction passing through
What shape did Rosalind Franklin propose based on X-ray crystalography evidence?
Spiral or helical molecule
Who reported that A=T and C=G?
Erwin Chargaff (created Chargaff’s rule)
How was the solution to the puzzle of the structure of DNA accelerated?
Model building
What does model building entail?
Assembly of 3D representations of possible molecular structures using known relative molecular dimensions and bond angles
Who used the model building technique to build a single, coherent DNA molecule?
Watson and Crick
What conclusions did Watson and crick use to build their model of DNA?
DNA is helical
DNA is antiparallel
What are the 4 key features that define DNA structure?
- Double stranded helix of uniform diameter
- Right handed
- Antiparallel
- Outer edges of the nitrogenous bases are exposed in the major and minor grooves
How are the two chains of DNA held together?
Hydrogen bonding between specifically paired based
How many hydrogen bonds form between adenine and thymine?
2
How many hydrogen bonds form between cytosine and guanine?
3
What is the pattern of pyrimidines pairing with purines known as?
Complementary base pairing
What does it mean that DNA molecules are antiparallel?
The 3 ‘ end pairs with the 5’ end of the other strand and vice versa
What chemical group is at the 5’ end?
OPO3-
What chemical group is at the 3’ end?
OH
What is the significance of base exposure in grooves?
Hydrogen bonded base pairs are accessible for potential hydrogen bonding
How are the surfaces of the AT and GC base pairs chemically distinct surfaces?
C=O group in T and N group in A are places for additional hydrogen bonding
Additional hydrogen bonding opportunities in GC
What is the significance of access to exposed base pairs in major and minor grooves?
Key to protein-DNA interactions in replication and expression
What are the 4 functions of DNA?
- Store genetic information
- Susceptible to mutation
- Precise replication
- Expression as the phenotype
What three substances did Arthur Kornberg show as needed to produce DNA with the same base composition as parental DNA in a test tube?
- Substrates deoxyribonucleoside triphosphates dATP, dCTP, dGTP and dTTP
- DNA polymerase enzyme
- DNA (template)
What were the three possible replication patterns that could occur in the experiment?
- Semiconservative replication
- Conservative replication
- Dispersive replication
What is semiconservative replication?
Each parent strand serves as a a a template for a new strand, two new DNA molecules each have one old and one new strand
What is conservative replication?
The original double helix serves as a template for, but does not contribute to, a new double helix
What is dispersive replication?
Fragments of the original DNA molecule serves as a template for assembling two new molecules, each containing old and new parts, perhaps at random
What type of replication did Watson and Crick’s original paper suggest?
Semiconservative replication
Who demonstrated that replication is semiconservative?
Meselson and Stahl
How did Meselson and Stahl distinguish old parent strands of DNA from newly copied ones?
Density labelling
What isotope did Meselson and Stahl use in their experiments do distinguish parent and copied DNA strands?
Heavy nitrogen (nitrogen-15): nonradioactive, heavy isotope
What bacterium did Meselson, Stahl and Jerome use in their experiments?
Escherichia coli
What two cultures of Escherichia coli did they grow?
One in nitrogen source was heavy Nitrogen
One in which nitrogen sources which was nitrogen 14 (light)
What was the nitrogen medium used in Meselson and Stahl’s experiments?
Ammonium chloride
How was the weight of the cultures determined?
Combined and centrifuged cultures, DNA samples were distinguished by different densities
How often did their bacteria cultures divide under the conditions used?
20 minutes
What happened when researchers transferred N-15 E.coli to normal N-14 medium and allowed it to continue growing?
They collected bacteria after each division
They extracted DNA from samples
What were the different density gradients found in each generation of the E.coli populations?
- Dense after transfer (uniformly N-15)
- After one generation, all DNA was intermediate density
- After two generations, two equally large DNA bands: one low density and one intermediate density
What was the density gradient in subsequent generations?
Proportion of low-density DNA increased steadily
What are the two steps of DNA replication?
- DNA double helix unwinds to separate template strands
- New nucleotides are joined by phosphodiester linkages to each new growing strand in sequence
To which end are nucleotides added to the growing new strand?
The 3 prime end (free -OH group)
What is the name of the huge protein complex the template strand interacts with to replicate DNA?
The replication complex
What is the purpose of the replication complex?
To catalyze the reactions involved
To what does the replication complex initially bind?
A base sequence called the origin of replication (ori)
In what ways does DNA replicate from the origin of replication?
In both directions, forming two replication forks
What is the binding of the replication complex to the ori a result of?
Recognition of different nucleotide bases by proteins
What part moves and what part remains stationary during DNA replication?
The replication complex remains stationary, DNA threads through
How does DNA enter and exit the replication complex?
Enters as a single strand
Exits as double strands
What do all replication complexes contain?
Several proteins with different roles in DNA replication
What is the first event at the origin of replication?
Localized unwinding (denaturation) of DNA
What forces hold the two strands together?
- Hydrogen bonding
- Hydrophobic interactions of the bases
What enzyme causes the DNA to unwind?
DNA helicase
What does DNA helicase use to make DNA unwind?
Energy from ATP hydrolysis
What binds to unwound DNA to keep unwound strands from re-associating?
Single-strand binding proteins
What is the purpose of unwinding DNA?
Makes the two template strands available for complementary base pairing
How many base pairs are there in small circular chromosomes such as those found in bacteria?
1-4 million
Where do small circular chromosomes replicate from?
A single origin
What happens as DNA from a small circular chromosome moves through the replication complex?
The replication forks grow around the circle, two interlocking circular DNAs are formed
How are the two interlocked DNA’s separated?
using enzyme DNA topoisomerase
How fast are DNA polymerases in E.coli?
1000 bases per second, 20-40 minutes for replication of all base pairs
How fast are DNA polymerases in humans?
50 bases per second- finished in an hour due to many polymerases working at many replication forks
Where are the origins of replication in large, linear chromosomes (such as human chromosomes)?
Many places
Adjacent ori’s can be bound at the same time
What shape is DNA polymerase?
Open right hand with a palm, thumb and fingers
How do DNA polymerases elongate polynucleotide strands?
By covalently linking new nucleotides to a previously existing strand
What is needed for DNA polymerase to being adding nucleotides to a chain?
A primer- a short single stand of RNA
What is the primer complementary to?
The DNA template strand
What synthesizes the primer?
An enzyme called primase
What do the ‘fingers’ of DNA polymerase do?
Recognize different shapes of the 4 nucleotide bases
How does primase work?
It binds to the template strand and synthesizes the primer, primase is released, DNA polymerase binds
What happens once DNA replication has been completed?
The primer is degraded
DNA is added in its place
Resulting DNA fragments are connected by enzyme action
What DNA polymerases are there in cells?
One is responsible for chromosomal DNA replication
The others are involved in primer removal and DNA repair
How many DNA polymerases have been identified in humans?
14,
Which DNA polymerase catalyzes replication in humans?
DNA polymerase 𝛿
What DNA polymerase catalyses replication in E.coli? How many are there?
DNA polymerase III
5
What name is given to the newly replicating strand?
The leading strand
What direction is the leading strand pointing in?
The right direction to grow continuously at its 3’ end
What name is given to the other strand (not the leading strand)
The lagging strand
What direction is the lagging strand pointing in?
The wrong direction- its 3’ end gets further and further from the fork
How is the lagging strand synthesized?
Small, discontinous stretches (100-200 nucleotides at a time, 1000-2000 in prokaryotes) from 5’ to 3’ end
What are the small, discontinous stretches of DNA on the lagging strand called?
Okazaki fragments
What does each Okazaki fragment require?
Its own primer
In bacteria, how does DNA polymerase III synthesize Okazaki fragments?
By adding nucleotides to a primer until it reaches the primer of the previous fragment
What happens when DNA polymerase III reaches the primer of the previous fragment?
DNA polymerase I removes the old primer and replaces it with DNA
What enzyme catalyzes the formation of the phosphodiester linkage between the Okazaki fragments after the primer is removed?
DNA ligase
What are DNA polymerases that makes them so fast?
Processive
What does it mean that DNA polymerases are processive?
They catalyze many polymerizations each time they bind to a DNA molecule
How is the newly replicated strand stabilized?
Using a sliding DNA clamp
What is the structure of a sliding DNA clamp?
Multiple identical subunits assembled into a doughnut shape
What does the DNA clamp do?
Binds to DNA just behind DNA polymerase, keeping it tightly associated with newly replicated DNA
What happens if the DNA clamp is absent?
DNA polymerase dissociates from DNA after 20-100 polymerizations
How many polymerizations can occur when the DNA clamp is present?
50,000
Why are telomeres not replicated?
When terminal primer of Okazaki fragments is removed, no DNA can be synthesized to replace it. New chromosome has a bit of single stranded DNA at each end
What happens when each new chromosome has a bit of single stranded DNA at each end?
Mechanisms cut off single stranded region along with some intact double stranded end
What do many eukaryotes have at the end of their chromosomes?
Repetitive sequences at the ends of chromosomes called telomeres
What is the telomere sequence in humans?
TTAGGG, repeated 2,500 times
What happens after 20-30 divisions in humans?
The chromosomes are unable to take part in cell division and the cell dies
How do constantly dividing cells such as bone marrow stem cells and gamete producing cells maintain telomeric DNA?
An enzyme, telomerase, catalyzes addition of lost telomeric sequences
Where is telomerase also expressed?
90% human cancers
What is the observed error rate in base replications in humans?
1 in 10^5 bases, 60,000 mutations for every human cell division
What are the three main DNA repair mechanisms that cells have?
- Proofreading mechanism
- Mismatch repair
- Excision repair
How does DNA polymerase perform a proofreading function?
It recognizes mispairing bases and removes the nucleotide and tries again
Lowers error rate to 1 in 10^10
What happens to repair DNA after DNA has been replicated?
A second set of proteins surveys the newly replicated molecule and looks for mismatches
How does mismatch repair mechanism detect wrong bases?
DNA strand is chemically modified after replication, methyl groups are added to adenines in prokaryotes. Unmethylated strands are therefore ‘marked’ and errors should be repaired
How is damage to DNA molecules repaired?
Excision repair
What does excision repair do?
Removes abnormal bases that have formed because of chemical damage and replaces them with functional groups
What is the name of the reaction that makes multiple copies of DNA?
The polymerase chain reaction
PCR reaction is_____, the sequence of steps is repeated over and over again.
Cyclic
What are the three steps of the PCR reaction?
- Double stranded fragments of DNA are separated into single strands by heating
- Short, artificial primer is added along with 4deoxyribonucleotide triphospates and DNA polymerase
- DNA polymerase catalyzes production of complementary strands
What is the exponential increase in the number of copies of DNA sequence called?
Amplifying sequence
What does the PCR technique require?
That the base sequences at the 3’ end are known so primer can be made
What bacterium lives in the hot springs of Yellowstone National park?
Thermus aquaticus
What temperature does Thermus aquaticus live up to?
95 degrees celcius
What technique allows researchers to determine base sequences?
DNA sequencing
What does DNA sequencing rely on?
Artificially altered nucleotides
What sugar replaces deoxyribose in nucleotide sequencing?
2,3-dioxyribose
What is the result of replacing deoxyribose with 2,3-dioxyribose?
dideoxyribonucleoside triphosphate
What happens when dideoxyribonucleoside triphosphate is added to the polynucleotide chain?
Lacks -OH group at 3’, next nucleotide is not added, synthesis stops
What are the steps of DNA sequencing?
- Denature
2. Mix with DNA polymerase, synthesized primers, 4 dNTP’s and ddNTP’s bonded to fluorescent tags
What happens after mixing denatured DNA to be sequenced with DNA polymerase, synthesized primers, 4 dNTP’s and ddNTP’s bonded to fluorescent tags?
DNA replication proceeds, test tube contains varying length synthesized strands, fragments are denatured from templates
What happens to the fragments that were denatured from templates?
Subject to electrophoresis
What does electrophoresis do?
Separates DNA fragments by length and detect differences as short as one base
What happens during electrophoresis?
Laser beam excites fluorescent tags- colour is detected and recorded. Length is fed into computer which prints DNA sequence of the fragment.
