Nucleic Acids and DNA Flashcards
Three key experiments to show that DNA is genetic material
- Griffith Experiment
- Avery, MacCleod, McCarty Experiment
- Hershey Chase Experiment (Bacteriophage T2and Radioisotopes)
The central dogma
1) DNA contains instructions
2) RNA transcribes DNA
3) proteins are made from the instructions
Frederick Griffith, 1928
Dr. Griffith described two strains of bacteria Streptococcus pneumoniae:
– lethal strain appeared smooth because it was surrounded by a polysaccharide coat
– non-lethal strain which does not have a polysaccharide coat, giving it a rough appearance.
Griffith’s experiment
Initially: Mice injected with the smooth strain would die, Those injected with therough strain would live.
Heat-killed S Strain mixed with R strain and the mouse dies. Even though the S strain was killed it
was able to “change” the R strain into a virulent strain
What was actually happening in griffith’s experiment?
S strain was heat killed, and the DNA and the outside coat fragmented. some of the DNA from the s strain entered the r strain. That piece of DNA is virulent, it holds the message for making the polysaccharide coat
Avery, Macleod and McCarty, 1944
Follow up on Griffith’s experiment.
– Demonstrated that this could be done in a test tube as well
We know that chromosomes are made up of protein and DNA (also knew about RNA in cells)
Treat the cells (S) with different enzymes to break down components and determine which has the transforming ability
– DNases - degrade DNA into individual subunits
– RNases - degrade RNA into individual subunits
– Proteases - degrade protein into individual subunits
Avery, Macleod and McCarty’s experiment
They added the enzymes to the killed S strain of bacteria, and then subsequently added it to the R strain. The tube with DNase was the only one on which the R strain wasn’t able to transform into the lethal S strain
Avery, Macleod and McCarty’s Conclusions
- DNase destroys ability of heated extract to transform Rstrain into lethal smooth (S) strain
- DNA must contain the transforming agent
- DNA must contain the genetic information
Radioisotopes
Elements are defined by the number of protons they possess
Electrons = protons in non-ionized forms #Neutrons can vary - this gives rise to isotopes
Radioisotopes that are commonly used in Molecular Biology
- P32- primarily used for labeling nucleic acids, can also be used for labeling proteins that have been phosphorylated
- S35- primarily used for labeling proteins
Detecting radioactive decay
Various particles are given off- The nucleus is unstable, and gives of particles to relieve the
tension
There are many detection methods
–geiger counters (detects what is being emitted into the air) typical of what you see in the movies - the beeping sound
– scintillation counters (radioactive emissions are converted to light)usually involves some sort of “swipe” and putting the cloth in scintillation fluid
– autoradiography (X-ray film) radioactivity exposes the film that is laid over
Viral genes entering a cell
1) Start of infection. Virus genes enter host cell. Protein coat does not.
2) Virus genes direct the production of new virus particles
3) End of infection. New generations of virus particles burst from host cell.
Hershey Chase Experiment (Bacteriophage T2 and Radioisotopes)
The experiment was to see if viral genes consist of DNA or protein (what’s the genetic material?)
In a virus, DNA was tagged with P32 and in a different set protein was tagged with S35, and both sets infected a culture of E. coli cells.the cultures were blended to separate the coats from the genes within bacterial cells. then they were it in a centrifuge, the bacterial cells settling at the bottom because they are heavier. In the tagged DNA set the radioactive DNA settled in the bottom with the bacterial cells wile the tagged protein was apparent in the solution with the coats.
Hershey Chase Experiment Conclusion
Viral genes consist of DNA. Viral coats consist of proteins.
Nucleotides
monomers that make up DNA
Nitrogenous base, sugar, phosphate
Other functions of nucleotides
Energy storage and transport
Ex: cyclic AMP, ATP, NADH
Polarity of a DNA Strand
- A DNA strand is a linear polymer of mononucleotides joined by phosphodiester bonds between the 3’ carbon of one nucleotide and the 5’ carbon of the next nucleotide.
- The bases are all joined to the 1’ carbons
Erwin Chargaff
showed that base composition (%)varies from organism to organism
Chargaff’s Rules: %A = %T, %G = %C
BUT: %A + %T did not equal the %G + %C
1952: Rosalind Franklin and Maurice Wilkins
performed X-RAY diffraction studies on DNA. Their datasuggested that DNA structure included:
A regular helix structure
Ladder-like rungs connecting parts of molecule
The Watson and Crick Model (double helix) accounted for:
X-ray diffraction pattern
Chargaff’s Rules
Also states the following:
Two-strands are antiparallel
Only two types of bp
Important Ramifications of theWatson-Crick Model
- Storage of genetic information
- Replication and inheritance
- Expression of thegenetic message
DNA Double Helix two important outcomes
- Gave rise to obvious and testable hypotheses about how genetic information could be stored
- Gave rise to the starting point for the development of techniques to study DNA
B DNA
Right handed , antiparallel, one turn is about 10.5 base pairs
A DNA
More condensed form of major/minor grooves
~11bp/turn more “tilt”
Z DNA
Easy in vitro, hard to find use in vivo. Bases flip outwards, can relieve stress
Left handed
Base Pairing in DNA
- A pairs with T
- G pairs with C
- An A-T base pair has two hydrogen bonds
- A G-C base pair has three hydrogen bonds
Charge of DNA
Negative
Bonds formed within and between strands
Hydrophobic stacking
Phosphodiester bond
Hydrogen bonding
Linear Representation of a DNA Sequence
5’-TGCCAGTTCAGCTAAACTCTG-3’
3’-ACGGTCAAGTCGATTTGAGAC-5’
- One strand is said to be complementary to the other and its sequence can be deduced from the base-pairing rules.
- Strands are anti-parallel
- A pairs with T and G pairs with C.
Base orientation: major vs minor grooves, protein bonding
CG differs from GC in major groove but not in minor groove. Order of bases determines how DNA interacts with specific proteins. Same goes for AT. Protein recognizes a promoter region. hydrogen bonding governs interactions. A= acceptor d= donator.
Denaturation/Renaturation of DNA
DNA can be “melted” with high temperature, and reannealed
Nuclear ion starts reannealment- 10-15 complimentary base pairs
Repeated fractions reanneal first
DNA melting curves
Increase in energy breaks the hydrogen bonds that stabilize DNA structure
Single-stranded DNA absorbs more UV light than double-stranded DNA
Changes in pH, ion concentration, and temperature can all denatureDNA
You can see how much DNA is denatured by how much light it is absorbing
How long is a strand of DNA?
3 meters
DNA Supercoiling
Supercoiling is the relaxing (negative supercoiling- relieves torsional strain, changes bond energy, easily denatured) and adding of twists(positive)
Tertiary structure
- Negative supercoiling
- Positive supercoiling
- It can occur in linear or circular chromosomes
When any portion of the circular DNA is loose supercoiling happens easily and naturally
Can happen in linear DNA as well
