DNA as the Genetic Material
Definition of genetic material - substance that carries the information determining the inherited properties of that organism. This information must be able to be transferred from parent to progeny.
Chromosomes were found to be the carrier of inheritance. But chromosomes are large aggregates of DNA and protein.
Classic experiment which led to identification of DNA as the genetic material. Originally scientists believed proteins. Nucleic acids only made up of four different types of nucleotides, thought not to be complicated enough.
Transformation experiments:
1928 - Griffith
Microbiologist working with Streptococcus pneumoniae</B>
Also known as: Pneumococcus (Fig. 3.6)
S strain - smooth edged colonies
virulence of strain associated with the polysaccharide coat or capsule
protects them from the immune response of the host
R strain - rough edged colonies
no polysaccharide capsule
non-pathogenic
Live S-----------------> Mouse dies
Live R-----------------> Mouse lives
Heat-killed S----------> Mouse lives
Heat-killed S+ live R---> Mouse dies
necropsied animals, only S strain bacteria found
some property of the dead S bacteria transformed the R
"the Transforming Principle"
Later it was demonstrated that heat killed S could be replaced by cell extract. Extract of S + live R-------------> Mouse dies
which molecule in heat-killed S transforming principle?
Avery, MacLeod, and McCarty, 1944
purified the "transforming principle" from extracts of S---it was DNA
also no effect with proteases or RNases, but did loose transformation activity when treated with DNases (proteases degrade protein, DNases degrade DNA)
Bacterial Transformation - Fragments of DNA penetrate the cell wall of the bacteria
Bacteria which are able to take up DNA in this manner are termed
competent
Bacterial cells can be treated with calcium chloride to make them more competent for recombinant DNA expermentation.
Another major experiment was performed by Hershey and Chase in 1944.
This experiment has come to be known as the Blender Experiment because a kitchen blender was used in the protocol.
The blender experiment utilized the bacteriophage T2 of E. coli.
One of the "T-even" phage, the T2 phage is a complex phage which contains a genome of ds DNA.
All viruses must contain a genome + a protein capsid. The T2 phage is a complex phage and also contains a tail sheath, base plate and tail fibers.
The capsid, sheath, base plate and tail fibers are all composed of protein.
At the time of Hershey and Chase it had not been definitively demonstrated that DNA was the genetic information. They designed the following experiment to proove that DNA was the genetic information for the T2 phage:
First the proteins of the phage were labeled with 35S-methionine/cysteine. The 35S radioisotope of Sulfur was incorporated into the methionine and cysteines of newly synthesized phage proteins. The phage proteins were the only molecules labeled.
In the first phase of the experiment, the 35S-labeled phage were allowed to attach to bacterial cells for a few minutes.
Un-attached phage were removed by a low speed centrifugation which resulted in a pellet of bacteria with attached phage.
The supernatant containing unattached phage was discarded.
The bacterial pellet was resuspended and a kitchen blender was used to shear off the attached phage.
The suspension was centrifuged, and the pellet and supernatant were both analyzed for amount of 35S label present.
Result:
80% of label in supernatant
20% of label associated with pellet
Interpretation: Majority of phage proteins remained in the supernatant, small quantity of protein associated with bacterial cells could be explained by residual tail fibers which were still attached to the bacterial cell walls following shearing.
DNA is the only phosphorous containing molecule associated with the phage. In the second phase of the experiment, the 32P radioactive isotope of phosphorus was used to radiolabel the DNA of the phage.
The steps above were then repeated.
Results:
30% of the label was in the supernatant
70% of the label was associated with the pellet
Interpretation:
DNA of phage was injected into bacterial cells and therefore, 70% of the total phage DNA was found associated with the pellet.
The 30% of label found in the supernatant could be explained by the presence of phage which had not yet injected DNA.
These results showed that DNA was the genetic information of the T2 phage and reinforced the idea that DNA was the "universal" genetic material.
Genetic material of all known living organisms DNA
viruses (which are infectious agents and non-living) may use DNA or RNA
Genetic information encoded in the sequence of the bases in nucleic acids
Required Properties of Genetic Material:
1) Ability to store genetic information and express it in the cell as needed.
2) Ability to transfer information to daughter cells with minimal error.
3) Physical and chemical stability.
4) Potential for heritable change without major loss of information.
Biological dogma: Genetic material always nucleic acid
But: Prions!
Theory: Some infectious diseases are caused by protein particles called prions and not by particles with nucleic acids like viruses, bacteria or eukaryotes. But how can they replicate, how can they increase in number and infect other organisms?
Thought to be infectious material that causes several degenerative neurological diseases like scrapie in sheep, mad-cow-disease and Creutzfeldt-Jakob disease in humans. No virus or bacterium ever found correlated with diseases.
Brain degenerates, animals cannot control body and act "mad".
Known: Prions are particles primarily of a cell-own protein that is misfolded. But they can interact with normally folded proteins and change them into misfolded ones.
Injected into brain they can cause disease. No nucleic acid found in prion particles!
Unknown: How they replicate and how they can infect healthy animals.