Griffith (1928)
(trying to see if DNA is the genetic material)
principles (2)
bacterial transformation
genetic recombination
bacterial transformation
genetic recombination by introduction of foreign DNA
genetic recombination
exchange of DNA between chromosomes
Diplococcus pneumoniae
wildtype: IIIS
virulent
smooth colonies
polysaccharide capsule
Diplococcus pneumoniae
mutant: IIR
non-virulent
rough colonies
no capsule
Griffith's transformation experiment: encapsulated
(define and outcome of injection into mouse)
smooth strain (virulent)
mouse dies
Griffith's transformation experiment: non-encapsulated
(define and outcome of injection into mouse)
rough strain (nonvirulent)
mouse lives
Griffith's transformation experiment: heat-killed smooth strain
(outcome of injection into mouse)
mouse lives
Griffith's transformation experiment: heat-killed smooth strain and rough straine
(outcome of injection into mouse)
mouse dies
What is the hypothesis (explanation) for outcomes?
genetic component transferred from Type IIIS to Type IIR
Avery, Macleod, and McCarty (1944)
critical methodologies (2)
selection (biological) for rare transformation
fractionation of cell-free extract to isolate single component
Preparation of transforming principle from S strain (steps)
begin with encapsulated strains
1. disrupt cells
2. centrifuge
now we have cell-free extract
1. precipitate with ethanol
2. redissolve in water
now we have transforming principle from S strain
Addition of transforming principle from S strain (steps)
transforming principle from S strain is added to test tube with R strain
mix and spread onto agar plates
a culture with both S and R cells form on plate
inject smooth strain (virulent)
mouse dies
inject rough strain (nonvirulent)
mouse lives
inject heat killed smooth strain
mouse lives
inject heat-killed smooth strain and rough strain
mouse dies
inject DNA from heat-killed smooth strain and rough strain
mouse dies
treat with ribonuclease (RNAse)
mouse dies
treat with protease
mouse dies
treat with nuclease (DNAse)
mouse lives
Hershey and Chase (1952)
"Blender Experiment"
used bacteriophage T2 to determine whether protein or DNA encodes genetic information
phage
protein head and tail with DNA molecules
inject DNA into bacteria
(proteins contain sulfer; DNA contains phosphates
Explain Hershey and Chase "Blender Experiment"
Testing Proteins
protein coats radiolabelled
bacteria infected
no radioactivity enters cell
phage grown with radioactive sulfur (35S)
centrifuge
radioactivity in supernatant
conclusion: proteins are NOT the genetic material
Explain Hershey and Chase "Blender Experiment"
Testing DNA
phage DNA radiolabelled
bacteria infected
radioactivity enters cells
phage grown with radiactive phosphorus (32P)
centrifuge
radioactivity in pellet
conclusion: DNA is the genetic material
minimal media
those that contain the minimum nutrients possible for organism growth, generally without the presence of amino acids, and are often used to grow "wild type" organisms
auxotrop
an organism unable to synthesize a particular organic compound (ex. amino acid) for its growth
selective media
used for selection of certain cells able to survive in selective media
Provide 2 examples of the use of minimal media
minimal medium lacking amino acids (thus lacking arginine) will select against N.crassa unable to synthesize it, and will select for "wild type" cells able to synthesize it
if an organism is resistant to a certain antibiotic (such as neomycin), then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing
Arg1
gene involved in Arginine synthesis
knock out Arg1
grow on minimal media
result?
no growth?
knock out Arg1
grow on minimal media supplemented with arginine
result?
growth
knock out Arg1
grow on minimal media supplemented with ornithine
result?
