Genetics: Lecture 4 Flashcards
History of DNA: Isolation, Structure and Replication
Who did the isolation of nuclein? When did this happen?
Friedrich Miescher is responsible for the isolation of nuclein in 1869.
What was Miescher’s question?
How to purify nuclein (nucleic acids) and extract them. Also wanted to determine their chemical composition.
Where did he get the nuclein from?
He got nuclein from white blood cells from the bandages of patients at a hospital (pus)
- Later on he also used salmon sperm for a nuclein source as it was rich with nuclein.
How did he purify the nuclein?
He used detergent and alcohol to purify the nuclein.
Findings on chemical composition?
Miescher established information on the chemical composition. It is made up of:
- hydrogen
- nitrogen
- oxygen
- phosphorous
- carbon
He also found that there is a unique ratio of phosphorous to nitrogen.
Main accomplishment or observation?
He isolated a new molecule from the nucleus – nuclein. This is what we now call DNA.
Who and when was the transformation of bacteria demonstrated?
Fred Griffith demonstrated the transformation of bacteria in 1928.
Question/Goal?
Griffith’s goal was to make a vaccine against Diplococcus pneumoniae, the causative agent of pneumonia.
- He wished to kill the deadly bacterium by heating to make a safe vaccine.
What did he use in his experiment?
He used a harmless R-strain of D. pneumoniae which produced rough shaped colonies on a petri plate.
He also used a virulent S-strain of the bacterium produced smooth shaped colonies on a petri plate (they are sugar coated and give protection).
What was his purpose?
He wanted to kill the pathogen by heat and inject it into humans top build their immune systems by making antibodies.
How did he do this?
Griffith injects live and heat-killed Diplococcus pneumonidae into mice. He performed 4 different experiments.
Griffith Experiment #1
He injected living S cells into the mouse (pathogenic control group). As a result, the mouse died.
Griffith Experiment #2
He injected living R cells into the mouse (nonpathogenic control). As a result, the mouse survived.
Griffith Experiment #3
He injected heat-killed S cells into the mouse (nonpathogenic control). The mouse lived as a result.
Griffith Experiment #4
He injected a mixture heat-killed S cells and living R cells into the mouse. As a result the mouse died. When it was looked at closer, there were living S cells found in the mouse.
Interpretation of Griffith’s experiment?
Q. Perhaps, the heat-killed S-cells in the mixture in Expt. #4 were not really dead?
A. Not true, because in Expt. #3, the mice injected with heat killed S-cells did not die.
Q. Perhaps, the R-cells in the mixture in Expt. #4 had mutated into the killer form?
A. Not true; Expt. #2 serves as a “control”.
Griffith’s conclusion?
- The ability to cause infection has been transferred from the dead S-cells to the harmless R-cells to make them virulent.
- The R-cells had been “Transformed”, i.e., a permanent change in the hereditary system of the R-strain of the bacterium coming from the S cells.
- But what is the transforming principle?
➢Protein or DNA? - the use of the word principle means that we have no idea what type of thing it is.
Who purified the transforming principle (DNA) and when did they do that?
In 1944, Oswald Avery purifies the transforming principle, DNA.
What did Avery do?
Avery repeated Griffith’s experiment, focusing on what the principle is, or further questioning the fourth experiment done by Griffith.
What was Avery’s question?
Avery’s questions was what is the principle that is transferred from dead S cells to R cells that makes them transform into S cells?
What was the main difference between Avery and Griffith’s experiments?
Avery did his experiment in-vitro (in class). This isolated variables and made them easier to control.
Avery first replicated/repeated Griffith’s experiment but added an extra step to separate out what was the principle?
What did Avery use in his experiments?
He used detergent to lyse the dead S cells.
What did he do in his experiment?
- He took lysate (liquid left after centrifuging) and mixed it with R cells and they became S cells. He knew the principle was there.
- He treated the lysate with enzymes to remove the sugar coat. When R cells were added, S cells still formed.
- He next added enzymes trypsin and chymotrypsin to remove proteins. When R cells were added, S cells were still formed.
- Next he employed Miescher’s technique to purify DNA by adding alcohol.
- Next, he got rid of RNA by adding enzyme RNAse. S cells were still formed altering adding R cells
- Finally, to prove his theory, they used DNAse to chew up the DNA. Finally, when R cells were added. No S cells were formed this time.
What was found in the lysate?
There are four components:
- Sugar coat of S cells
- Protein
- RNA
- DNA
Avery’s conclusion?
Avery concluded that the transforming principle was DNA.
What are viruses composed of?
Viruses are composed of only nucleic acid (DNA and RNA) and proteins.
What are phages? Bacteriophage?
Phages are viruses that attack vertebrates and mammals.
Bacteriophage are viruses that attack bacteria. They land on bacteria and inject their DNA. The injected DNA creates more phage and a bunch more is freed when the bacteria bursts open. Then they infect others.
Hershey and Chase’s Questions?
They wanted to determine if it was the DNA or the proteins that was infecting one another.
- Differentiate proteins from nucleic acids.
When was the Hershey-Chase Experiment?
1952
Theoretical Basis of Hershey-Chase Experiment?
- Protein contains sulphur, but lacks phosphorous. Protein can be specifically labelled with radioactive sulphur (S35)
- DNA contains phosphorous, but
lacks sulphur. DNA can be specifically labelled with radioactive phosphorous (P32). - By this method, either DNA or protein can be detected in cells.
Hershey-Chase Experiment? Batch 1
Batch 1: Phages were grown with radioactive sulphur (s35), which was incorporated into phage protein.
- Mixed radioactively labelled phages with bacteria. The phages infected the bacterial cells.
- Agitated the mixture in a blender to free phage parts outside the bacteria from the cells.
- Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube; free phages and phage parts, which are lighter, remained suspended in the liquid.
- Measured the radioactivity in the pellet at the bottom and the liquid at the top.
Conclusion: Whatever makes more phage is not protein.
Hershey-Chase Experiment? Batch 2
Batch 2: Phages were grown with radioactive phosphorous (P32), which was incorporated into phage DNA.
- The same exact method was done.
- Mixed radioactively labelled phages with bacteria. The phages infected the bacterial cells.
- Agitated the mixture in a blender to free phage parts outside the bacteria from the cells.
- Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube; free phages and phage parts, which are lighter, remained suspended in the liquid.
- Measured the radioactivity in the pellet at the bottom and the liquid at the top.
The capsids are not radioactive.
Conclusion: Only bacterial DNA injected by the phage is radioactive. This determined that DNA = genes and were the infectious material.
Chemical Structure of DNA ?
DNA is composed of a double helix, arranged in an antiparallel arrangement.
