Genetic Lecture 4 Flashcards
Order of experiments for the discovery of DNA?
Isolation of nucleic, transformation of bacteria, purification of DNA: the transforming principle, radioactive DNA, X-ray diffraction, DNA model, semi-conservative DNA replication, and molecular components of DNA replication
Freidrich Miescher
Wanted to become a doctor but had troubles hearing so he became a scientist (1869). Scientists at this time were still debating the concept of the cell.
What was Freidrich Miescher Question?
What’s in the nucleus?
What was Freidrich Miescher experiment?
His supervisor told him to study nuclein and told him he could find it in white blood cells. So he collected bandages and scraped pus off of it. Then he put it in a tube and purified the nuclein by using alcohol and detergent (a method still used today).
What was Freidrich Miescher results?
Isolated a new molecule from the nucleus – nuclein (now known as DNA). He came up with the chemical composition of nuclear acids. Made up of hydrogen, oxygen, nitrogen and
phosphorus. Unique ratio of phosphorus to nitrogen. He also found that salmon sperm was a fish source of nuclein. However, he didn’t know the importance of nuclein.
Fred Griffith
Griffith wanted 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. By doing this he wanted to kill pathogens by heat and put in the vaccine so when the actual thing comes you already have antibodies for it (1928).
What are the two strains of bacteria that Griffith used?
A harmless R-strain of D. pneumoniae produced rough shaped colonies on a petri plate. R stands for rough and it doesn’t have a sugar coat. A virulent S-strain of the bacterium produced smooth shaped colonies on a petri plate. S stands for the sugar coat it forms.
What was Griffith’s experiment?
Griffith injects live and heat- killed
Diplococcus pneumoniae into mice. #1 Living S cells = mouse dies. #2 Living R cells= mouse healthy. #3 Heat-killed S cells= mouse health. #4 mixture of heat killed S cells and living R cells=mouse dies but living S cells are present.
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”.
Conclusion from Griffith’s Experiment?
The ability to cause infection has been transferred from the dead S-cells to the harmless R-cells. The R-cells had been “Transformed”, i.e., a permanent change in the hereditary system of the R-strain of the
bacterium. Something transferred from the dead S cells to harmless R cells that makes them pathogenic/virulent. But he didn’t know if the transforming principle was protein or DNA.
Transformation?
The transfer of exogenous DNA into a host cell, thereby causing a permanent change in the hereditary system of the bacteria.
What was Oswald Avery’s experiment?
He repeated Griffith’s 4th experiment. However, instead he did his experiment in vitro. He added detergent to heated s cells and isolated the lysate (centerfige) then mixed lysate with the R cells, and it transformed the R cells into S cells.
In lysate we have many different things: protein, DNA, RNA, and the sugar coat. To figure out the principle you just eliminate things one by one. First enzyme chews up the coat sugars, then mix with the R cells again. Repeat until it’s just the DNA and RNA. Then purify them with alcohol, then put into water. Then destroyed RNA by RNase. DNA still results in S cells, then remove it and get R cells.
In Vitro
In glass. It’s easier to control the variables and isolate the parts needed.
In Vito
In animal/ living thing.
Avery’s Results (1944)?
Figured out it was DNA bringing the info from one generation to the next. However, some people still thought the DNA was doing something to the R cells turning them on and off. Because it’s not complex enough to be doing all of that. Thought it was protein that was the principle.
What strain of DNA is infectious?
The S strain because it has the sugar coat to protect it from the host’s immune system.
What did Avery mix with the R cells?
Lysate
What are viruses composed of?
Nucleic Acid (DNA OR RNA) and Protein
What did Hershey and Chase use in their Experiment?
Using a virus called bacteriophage that attacks bacteria. It’s made of a capsule and they land on bacteria and inject their DNA into the bacteria. Then it starts to make more of the virus and then the bacteria bursts open, freeing a whole bunch of phage. It’s a chain reaction.
How did they differentiate protein and DNA?
Proteins have suppler from the dicysteine bonds which have cystene which have sulphur. They used an isotope of sulphur that’s radioactive. Incubated phage with bacteria and grew them with S35, felt is phage where all of its proteins had some of this isotope. Did the same thing with DNA and phosphate.
Hershey-Chase Experiment (1952):
The Theoretical Basis?
Protein contains sulphur, but lacks phosphorous. Protein can be specifically labelled with radioactive sulphur (35S), can then be detected with a machine (scintillation counters). DNA contains phosphorous, but lacks sulphur. DNA can be specifically labelled with
radioactive phosphorous ( 32P). By this method, either DNA or protein can be detected in cells.
Hershey-Chase Experiment?
Phages were grown with radioactive sulphur, which was incorporated into phage protein (pink). Mixed radioactively labelled phages with bacteria. The phages inflected 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 and liquid. No radioactivity in the bacteria (bottom). Repeated with phages grown with radioactive phosphorus which was incorporated into phage DNA (blue). The pellet was found to be radioactive.
Hershey-Chase Results?
That it is DNA that is transferring the information, not protein.
After the 4 experiments people knew what?
That it’s DNA that carried to info, so now they wanted to know its structure.
What’s a nucleotide?
The building block of DNA. Has five carbon deoxyribose sugar group. A phosphate group, and a nitrogenous base. They are joined together to form a polymer of a DNA strand.
The backbone of DNA consists of what?
A succession of sugars and phosphates.
Why is it important where the carbons in DNA are?
It gives directionality to DNA.
What is on the important carbons of DNA?
1’C has the nitrogenous base
3’C has the OH group
5’C has the phosphate group on it
What are the base pairs?
A and T/U
G and C
Strands of the DNA molecular are what?
Anti-parallel:
5’»»»»»»>3’
3’««««««<5’
Why is it called nitrogenous bases?
Because there is a lot of nitrogen in the bases.
