DNA as genetic material experimental proof: Hershey and chase experiment

In the early 1950s, there were scientists who still debated whether protein or DNA was the genetic material in cells. To settle the controversy, in 1952 Alfred Hershey and Martha Chase carried out a series of experiments to determine the genetic material in T2 phage (whether it is the phage protein or the phage DNA was transmitted in phage reproduction). Finally Hershey and chase concluded that DNA, not protein function as the T2 phage genetic material.

Bacteriophages (viruses that affect bacteria) were the key element for Hershey and Chase experiment.

Example: T2 phage (Host bacterium: E .coli)

 Structure: Tad pole like structure

T2 phages consist of approximately 50 percent protein and 50 percent DNA.

DNA: Linear and double stranded (enclosed within the capsid)

It has a polyhedral head connected to a helical tail through a short collar.

It also contains hexagonal base plate and tail fibers.

Figure 1: Structure of T2 bacteriophage

Reproduction cycle of T2 phage

The virus doesn’t have their own mechanism of reproduction but they depend on a host for the same.

(Here in case of bacteriophages, bacteria are their host)

The production of new viruses occurs within the bacterial cell.

  • Infection is initiated by adsorption of the phage by its tail fibers to the bacterial cell.
  • Once they attach to the host cell, each virus acts as a molecular syringe by injecting its genetic material into a bacterium. The empty viral capsule remains attached to the outside of the cell.
  • The entry of a viral component changes the genetic program of the host bacterial cell: it is converted from a bacterium into a bacteriophage factory.

(The bacterial cell starts to replicates the viral component and the viral component “commandeers” the cellular machinery of the host to synthesize viral proteins).

  • Then the phage DNA becomes encapsulated within the proteins, producing progeny phages.
  • About 20 minutes later, the bacterial cell lyses, releasing dozens of viruses
  • This process is referred to as the lytic cycle.
Figure 2: Life cycle of T2 bacteriophage (lytic cycle)

Hershey and Chase conducted a series of experiment to discover whether it is protein or DNA that is injected into the E.coli during phage infection and which one is used as the genetic material to build new phages?

To trace the two components (protein and DNA) of the virus over its life cycle, Hershey and Chase labeled each with a specific radioactive tracer:

They used radioactive forms (isotopes) of phosphorus and sulfur.

Radioactive isotope of phosphorus: 32P

Radioactive isotope of sulfur: 35S

A radioactive isotope can be used as a tracer to identify the location of a specific molecule, because any molecule containing the isotope will be radioactive and therefore easily detected.

 Reason for choosing sulfur and phosphorus for labelling

  • DNA is the only phosphorus containing substance in the phage particle. The deoxyribose phosphate “backbone” of DNA is rich in phosphorus, but this element is not present in most proteins.
  • Viral proteins contain some sulfur (in the amino acids cysteine and methionine), but it is absent in DNA.

Such differential labelling would enable one to distinguish between DNA and proteins of the phage without performing any chemical tests

Hershey – Chase Experiment

 This will be explained step by step so that you never get confused.

 Step 1: Labelling of T2 phage with radioactive isotopes

 Batch 1:

  • Hershey and Chase grew a batch of T2 bacteriophage in E.coli culture in the presence of 35S.

(That means at first Hershey and Chase grown E. coli in a medium containing 32P and infected the bacteria with T2)

The resulting progeny viruses had their proteins labeled with this 35S.

Batch 2:

  • The researchers grew another batch of T2 bacteriophage in E.coli culture in the presence of 32P.

The resulting progeny viruses had their DNA labelled with 32P.

Figure 3: Labelling of T2 phage with radioactive isotopes

 Step 2: Infection

Experiment 1: Phages labeled with 35S were added to an unlabeled culture of E. coli

Experiment 2: Phages labeled with 32P were added to an unlabeled culture of E. coli

Then sufficient time was allowed for the phages to infect the bacterial cells.

Figure 4: Infection of unlabelled E.coli culture with 32P and 35S labelled phages

Step 3: Blending

After a few minutes, they agitated the mixtures vigorously in a kitchen blender, which stripped away the parts of the virus that had not penetrated the bacteria.

This rough treatment neither injured the bacteria nor altered the course of the phage infection.

(This experiment known as the blender experiment because a kitchen blender was used as a major piece of apparatus)

 Step 4: Centrifugation

The mixture is centrifuged in a test tube to separate the bacterial cells from the virus (phage ghost).

The centrifuge separated the lighter phage particle from heavier bacterial cells.

Phage ghost: Empty protein coat (no DNA)

After centrifugation they got 2 parts in the test tube: Pellet (bacterial cell) + Supernatant (virus particle)

Step 5: Radioactivity measurement

Radioactivity in the pellet and supernatant (liquid) is measured.

Result of experiment 1: Radioactivity detected in the supernatant (Radioactivity by 35S)

Result of experiment 2: Radioactivity detected in the pellet (Radioactivity by 32P)

Most of the 32P-labeled DNA had entered the bacterial cells, while most of the 35S-labeled proteins remained in solution with the spent viral particles.

Conclusion: When the bacterial virus (bacteriophage) T2 infects its host cell Escherichia coli, it is the phosphorus-containing DNA of the viral particle, not the sulfur-containing protein of the viral coat, that enters the host cell and furnishes the genetic information for viral replication.

Figure 5: Seperation of phage ghost from bacterial cell and radioactivity measurement of pellet and supernatant

Step 6: Culturing of phage infected bacterial cell

  • Phage infected bacterial cells from both experiments was cultured in an appropriate medium.
  • Eventually, the cells burst and new phage particles emerged.
  • After that phage progeny was studied for radioactivity.
Figure 6: Culturing of phage infected bacterial cell

Observation of experiment 1: When new phages emerged from the cell, they contained almost no radioactivity

 Conclusion: Although the protein component of a phage was necessary for infection, it didn’t enter the cell and was not transmitted to progeny phages

 Observation of experiment 2: Many of the new progeny phages emitted radioactivity from 32P

 Conclusion: DNA from the infecting phages had been passed on to the progeny. DNA entering the host cell carries all the genetic information for the synthesis of new phage particles

These results of Hershey and Chase experiment proved that DNA, not protein, is the genetic material of phages.

Figure 7: Summary of the Hershey – Chase experiment demonstrating that DNA, and not protein, is responsible for directing the reproduction of T2 phage during the infection of E.coli

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