DNA story (8): Hershey-Chase experiment, Isotopic Labelled Phage infects E. coli

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Background of Hershey-Chase experiment

Although Oswald Avery had already proven the transforming principle of bacteria was DNA 8 years ago, Hotchkiss had even reduced the protein content to 0.02%, yet nearly all scientists believed that protein couldn’t be ruled out as genetic material completely. Meanwhile, Max Delbruck hoped to apply the principle of complementarity in quantum mechanics to life phenomena, particularly Bohr's lecture on light and life inspired and prompted him to transition from physicist to biologist. At Caltech, Max Delbruck created phage group with a free academic style like the Copenhagen School to attract numerous biologists and physicists who were interested in uncovering the fundamental laws of life. Among them was Hershey, one of the founders of molecular biology.

Before the Hershey-Chase experiment, scientists already knew that phages were viruses that infected bacteria. Phages were composed solely of a protein coat and internal genetic material, DNA. Their extremely simple structure and rapid reproduction made them very suitable as model organisms for biological experiments. During infection, their lower appendages grasp the bacteria to inject hereditary material like a syringe. After many replications, the bacteria burst to release progeny phages. Since it was still unknown whether the injected substance contained protein, Hershey and Chase decided to use radioactive isotopes to label phages. Isotopic labeling is a new technology that allowed scientists to accurately trace the molecules in biochemical reactions.

Hershey-Chase experiment or waring blender experiment

Almost all biology books have transformed or distorted the Hershey-Chase experiment in order to make it more understandable and convincing. For example, it is described as a single experiment, but in fact, it was a collection of several experiments. In one of experiments, Hershey and Chase only measured the radioactivity of solution, yet it is embellished that precipitate was highly radioactive. Huge experimental errors are ignored, so the experimental results are seemingly taken for granted. We try to describe the experiment as closely as possible to its original form, while keeping it accessible.

They first cultured E. coli in petri dishes containing radioactive elements, then added phages, because virus metabolism must rely on host. Radioactive sulfur, ³⁵S, could only label the protein coat, as all sulphur was concentrated in the protein. Likewise, radioactive phosphorus, ³²P , in another petri dish could only label DNA. Their first experiment replicated and developed the results of Thomas Anderson via radioactive isotope labeling: when salt concentration changed drastically, phages released their DNA and left behind DNA-free protein coat; the protein coat easily attaches to bacteria, but DNA does not; if the phages adsorbed onto killed bacteria or bacterial debris, the DNA could easily be digested by enzymes. Thus, the protein coat would protect DNA from hydrolysis of DNase.

In the following experiment, E. coli was infected with phages labeled with radioactive ³²P. Then the culture fluid was stirred with a Waring blender for 0~8 minutes to remove protein coat attached to the surface of bacteria. Waring blender preserved the integrity of bacteria and phage genetic material that had entered bacteria. So it had another name waring blender experiment. The liquid and bacteria were separated in a centrifuge. Hershey and Chase detected only about 21–35% of ³²P in the supernatant. This suggested that much of DNA had entered bacteria, but it could not exclude the possibility that protein was injected in bacteria. So, Hershey and Chase repeated the experiment with phages labeled with radioactive sulfur. This time the result was opposite: 80% of ³⁵S was in the supernatant. Most of protein had not been injected into bacteria but was merely attached to their surface.

However, the Waring blender only removed 80% ³⁵S-labeled protein coat. To prove the missing 20% didn’t replicate in bacterial cells, Hershey and Chase conducted another experiment. This time, they still used Waring blender to remove protein coat, but phages were allowed to reproduce for one generation. Ultimately, only 1% radioactive sulfur and 30% radioactive phosphorus were detected in the precipitate ( progeny phage).

Frequently Asked Questions

Why was Hershey-Chase experiment accepted by people?

Oswald Avery's experiment had only been contaminated by less than 0.02% of protein, whereas the protein that interfered with experiment of Hershey and Chase was about 1%. Even the whereabouts of 20% protein was not be explained. Therefore, Hershey and Chase could only conclude that DNA might be hereditary material. However, it is surprising their experiment was quickly and widely accepted by scientists. The debate about hereditary material was completely ended.

Oswald Avery needed to challenge the tetranucleotide hypothesis prevailing for 30 years, so his resistance was greater than anyone. But 8 years later, the status of protein as the primary genetic material had already been severely shaken: Hotchkiss found that bacteria could acquire penicillin resistance through a transforming substance; improvements in nucleic acid extraction enabled other scientists to obtain large amount of purer DNA, and they detected that DNA was present in equal amounts in all cells of the same species, but reproductive cells had only half. Erwin Chargaff used new techniques to prove tetranucleotide hypothesis was incorrect, as A=T, G=C, but A≠G.

Compared to Avery's pneumococcus transformation experiment, the Hershey-Chase experiment also had several advantages. It was highly repeatable and easily operated, whereas DNA extraction was very difficult at that time. The transformation principle only changed one characteristic of pneumococcus; however, the invading DNA resulted in reproduction of complete viruses. Phages infection could be intuitively depicted: they injected DNA like a syringe, and left protein coat on bacteria surface. Overall, Hershey-Chase experiment was the straw that broke camel's back.

Anec  > Biology > Genetic material

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