Lecture 21 Flashcards
Friedrick Miescher
- first isolate DNA from white blood cells found in pus on bandages
- called it nuclein
- noted it was acidic and high in phos
Albrecht Kossel
- characterized DNA
- isolated the bases
- determined DNA has 4 nitrogenous bases
Phoebus Levene
- DNA is made of nucleotides
- each nucleotide has a phosphate, base, and sugar
Tetranucleotide Structure
- proposed as the structure of DNA by Levene
- accepted as corrected for years as there was little interest in DNA by geneticists at the time
What was originially thought to be the genetic material? Why?
- protein
- very abundant - 50% of cell dry weight
- variable
- tetranucletide structure too simple to account for variability
- protein was accepted without being tested as most researchers still interested in transmission genetics at the time
Describe steps of Griffeth’s experiment
- studied streptococcus pneumoniae in mice
- smooth strain kills mice, rough strain does not
- heat killed smooth strain and injected in mouse - the mouse lived
- heat killed smooth and mixed with live rough then injected in mouse - the mouse died
What did Griffeth’s experiment show?
- something from the dead smooth bacteria transferred to the live rough bacteria to make them become virulent
- he coined this transformation
transforming principle
the term Griffeth gave to the agent that causes transformation
note he did not know what the agent was
characteristics of the transforming principle used to help identify the principle
- transformation can occur in a test tube
- transformation can be accomplished by incubating filtrate from heat killed cell with nonvirulent cells showing that intact virulent cells are not required
Avery, McCarty, and MacLeod
- identified the transforming principle
- filtrated heat killed smooth s. pneumonia
- divided between 3 tubes and treated one with Dnase, Rnase, and protease
- looked for the presence of transformation in each tube
- transformation occurred in the tubes treated with Rnase and Protease but not Dnase proving that DNA is the transforming principle
Who discovered the identity of the transforming principle?
Avery, Mccarty, and Macleod
Who discovered transformation?
Griffeth
Hershey and Chase
- DNA is the genetic agent in bacteriophages
- labeled the DNA in a T2 bacteriophage with 32P and the protein with 35S
- placed in separate flasks and allowed the viruses to infect bacteria
then separated bacteria from anything attached to them - looked to see if the bacteria were radioactive and also allowed infected bacteria to grow on a plate to see if phage reproduction occurred
- Phage reproduction occurred in both cases
- Bacterial cells only became radioactive when 32P was used
What did Hershey and Chase show?
- only the DNA from the virus went into the bacteria, the protein did not
- phage replication occurs regardless of what gets into the cell
- therefore, the DNA is the infectious agent of the bacteriophage
Why did Hershey and Chase use the isotopes they did?
- needed to distinguish the two molecules
- protein does not have Phos and would not be marked by the 32P while DNA does
- DNA does not have sulfur and would not be marked by the 35S while protein does
Fraenkel-Conrat and Singer
- RNA is the genetic material in RNA virus
- utilized tobacco mosaic virus
Yanofsky
- colinerarity exists between DNA and protein
- studied tryptophan synthetase genes and proteins using a deletion map and found that the order of the point mutations on the DNA was the same as the altered amino acids in the protein
Wilkins and Franklin
- x-ray diffraction studies to try to determine the structure of DNA
- found helical structure with two other repeating structures
Values discovered by Wilkins and Franklin
- the helical structure of DNA has a constant width of 2 nm
- repeating structure every 0.34 nm and every 3.4 nm
10 angstroms =
1 nm
Chargaff
- DNA base composition studies
- compared percentages of different bases
- determined…
- the number of purines is equal to the number of pyrimides
- the number of Adenines is equal to that of Thymine
- the number of cysteines is equal to that of guanine
How did the discoveries of Chargoff, Franklin, and Wilkins influence Watson and Crick’s model?
- figured out that a purine and pyrimidine must be paired to maintain the constant width size discovered by Wilkins and Franklin
- paired and A with T and C with G to maintain the ratios determined by Chargoff
Who was awarded the Nobel prize for determining the structure of DNA?
Watson, Crick, and Wilkins
component of a nucleotide
nitrogenous base
phosphate
sugar
what is the charge of phosphate?
negative
sugar in DNA
deoxyribose
sugar in RNA
ribose
difference between ribose and deoxyribose
- ribose has a hydroxyl group at the 2’ carbon
- deoxyribose has only a hydrogen at the 2’ carbon
What are the bases in DNA
adenine
thymine
guanine
cytosine
What are the bases in RNA
adenine
uracil
guanine
cytosine
Name the purines
adenine
guanine
Name the pyrimidines
cytosine
thymine
uracil
double ring structure
purine
single ring structure
pyrimidine
nucleoside
sugar plus a base
nucleotide
sugar plus a base plus a phosphate
another name for a nucleotide
nucleoside monophosphate
nucleoside with the addition of 1 phosphate
what is a nucleotide triphosphate
- seen in replication
- a sugar, base, and 3 phosphates
What is connected to the 1’ carbon of the sugar in a nucleotide
the base
What is connected to the 5’ carbon of the sugar in a nucleotide
the phosphate
The are ___ bonds between adenine and thymine
2 hydrogen
There are ___ bonds between guanine and cytosine
3 hydrogen
hydrogen bonds
relatively weak attractions between positively and negatively charged molecules
What is the main force that joins 2 strands of DNA
the hydrophobic interactions of the stacked bases in a double helix
What allows a constant helix width
complementary base pairing between 2 strands of nucleotides
What role does complementary base pairing play in the fulfilling Chargaff’s data on base composition?
maintains the equal ratio of A:T, C:G, and A+G:T+C
What role does complementary base pairing play in fulfilling Franklin and Wilkin’s data X-ray diffraction data?
maintains the constant 2nm width of the double helix
phosphodiester bond
- joins the sugar-phosphate backbone
- attaches the 3’ of one nucleotide to the 5’ of the next allowing them to align properly with the opposite strand
antiparallel
run in opposite directions
gets the bases in the proper order to align with each other
pletonic coil
strands have to be unwound to separate
hydrophobic interactions
- DNA backbone is hydrophilic
- bases are hydrophobic
- this pushes the backbone to the outside and the bases to the inside
- these interactions are beween adjacent base pairs and form the sstabilizing force of the double helix
main force in stabilizing the double helix
hydrophobic interactions
form the specificity of the DNA strand interactions
hydrogen bonding
major and minor grooves
formed by the stacking of base pair
each turn of a helix contains 1 major and 1 minor groove
what maximized hydrophobic interactions?
base stacking
Watson Crick model
right handed
10 bp/turn
A-DNA
more compact that W-C “B-DNA” model - 11bp/turn
still right handed
Z-DNA
zig zag DNA
left-handed coil
max absorption of UV by DNA occurs at…
a wavelength of 260 nm
UV absorption
- the presence of DNA in a sample is detected by observing the UV absorption of the same at 260
- a correction is then done to quantify DNA from proteins/other molecules that also absorb UV at 260 nm
- absorption increased by 40% when DNA is denatured
centrifugation
separate nucleic acid fragments
heavier materials go to bottom
velocity centrifugation
- sucrose gradient
- measure speed at a which a molecule moves to the bottom of the tube
- Svedberg units (S)
- in general, greater mass results in greater speed but shape also influences speed
density gradient centrifugation
- equilibrium centrifugation
- cesium chloride base medium
- molecule migrate through until the reach neutral buoyant density
isopycnic point
point of neutral density which molecules migrate to in density gradient centrifugation
Is GC-rich DNA or A-T rich more dense
GC - it will end up at the bottom of the tube in density gradient centrifugation
What do we know about the nucleotides in the DNA that goes to the bottom of the tube in density gradient centrifugation?
It has the highest G-C content
DNA has a ___ charge because…
negative
phosphate is negative
In electrophoresis, DNA fragments move towards…
the positive pole
Will a larger or smaller DNA fragment move further through the gel in electrophoresis
smaller
Why are controls run in gel electrophoresis?
can determine the sizes of sample fragment by comparing their migrations to that of the control fragments
- use a mathematical relationship
describe electrophoresis
- used to separate DNA fragments based on differenced in size as they migrate through an electrical current
denature the DNA
breaking the hydrogen bonds and hydrophobic interactions which causes the two strands to separate
can be used to characterize the DNA
how can denaturation of DNA be accomplished
heat
high pH
hyperchromic effect
increased UV absorption by DNA due to denaturation
Tm
midpoint of thermal denaturation
point at which 50% of DNA is denatured
melting curve
- observe the effect of heating DNA
- relative absorption over a range of temperatures
- sigmoidal curve
- show Tm and possible molecular configurations for various stages of melting
High ___ content results in higher Tm
G-C
Tm =
69 + (.41)(%GC)
