Background & Introduction

Paper Title: Independent functions of viral protein and nucleic acid in growth of bacteriophage 
 

Alfred Hershey (1908-1997) and Martha Chase (1927-2003)

Series of experiments to determine the role of protein and DNA in bacteriophage

What is known at this point in time:

1848: Chromosomes discovered; exchanged during mitosis

                Wilhelm Hofmeister: cell nuclei resolve into small, rod-like bodies during mitosis

1869: DNA isolated from cells

                 Friedrich Miescher: nuclei contain a phosphorous-rich molecule, "nuclein"

Early 20th century:

1902 Walter Sutton hypothesizes that chromosomes are hereditary units

          Chromosomes carry genes; basic units of heredity

          Genes are arranged linearly on chromosome 

          Chromosomes are made of both protein and DNA - didn't know which one carries information

Many thought proteins were better candidates; 20 amino acids vs 4 nucleic acids

Life is complicated, DNA is a relatively simple molecule

1928: Griffith’s Experiment showed Genes were able to be transferred

1944: Avery, MacLeod and McCarthy: transformation only occurs when DNA was present, and occurs when proteins are removed

1952: Oversimplification of the Hershey and Chase experiment as portrayed in a textbook. 

Methods:

Phage DNA and Protein can be detected using radiolabelled isotopes:  P³² and S³⁵  

Phosphorus containing DNA labelled with P³² 

Sulfur containing proteins labelled with S³⁵

Preparation of radiolabelled phage

Results:

Lead up experiments 

Experiments 1 and 2:

Ghosts are empty phage particles adsorbed to bacteria

Ghosts can be created by plasmolysis

DNA becomes acid soluble when fragmented by DNase

Protein is not acid soluble

Using radiolabelled isotopes, characteristics of phage DNA and protein can be observed (Table 1)

DNA is in the supernatant while protein stays in sediment (Table 2)

Experiments 3 and 4:

Adsorption of phage to a bacterium is followed by the release of DNA from the protein coat 

Shown through the use of DNase on frozen, thawed, and fixed bacterial cells after infection (Table 3)

Followed by tests involving addition of phage to bacterial debris (Table 4)

Experiment 5:

The blender experiment indicates that protein does not enter into the cell, while DNA does (Figure 1 )

Preformed by allowing phages to infect and using a blender to break capsids off of cells 

Possible due to "precarious attachment" of phage to bacteria

Experiment 6:

If more time is allowed for infection and replication of the phage it has no effect on the amount of S³⁵ sedimented (Table 6)

Showing that there is little S³⁵going into phage progeny. 

Did a similar experiment with P³² and about 30% of it was in phage progeny

S³⁵ was only 1% in phage progeny.

Experiment 8:

Fixing the DNA into the phage with formaldehyde affects the replication of phage (Table 8)

Much lower plaque titer (1000 fold decrease)