As seen in the last post, Avery hypothesized that the Transforming principle of Griffith’s experiment could be DNA. This hypothesis was supported as well as criticised at the same time. It was criticised as most of the scientists promoted protein as the genetic material.
At around the same time, Max Delbrück and team was working on the use of bacteriophage viruses in biology. The three important researchers of this group were Delbrück, Salvador Luria and Alfred Hershey. They were unsure of Avery’s hypothesis initially.
However, later, following the experiments conducted by Alfred Hershey and Martha Chase, they rejected protein and supported DNA as the hereditary material. They published their work titled ‘Independent functions of viral protein and nuclei acid in growth of bacteriophage’ in 1952.
Let’s have a look at the Hershey and Chase’s experiment:
Hershey with his colleague, Martha Chase, began a series of experiments to identify the functions of protein and DNA in bacteriophage. They worked with T2 phages (fig 2) and host E.coli for the same.
They radiolabelled the DNA and the protein (of coat) with radioactive isotopes of S (S35) and P (P32), respectively. For radiolabelling, they cultured E.coli in the media with radiolabelled molecules. These radiolabelled E. coli were then infected with the phages, which in turn got labelled. These radiolabelled phages were then used to infect the unlabelled E. coli (see fig 3). After proper time period, the bacteria (heavier) and bacteriophage (lighter) were separated using the centrifuge, at 2000G and 12000 G respectively. On centrifuging at 2000G, the bacteria sedimented and the bacteriophages (viable and ghost) remained in the supernatant. The radioactivity was measured using end-window Gieger counter.
Through first sets of experiments, Hershey and Chase seconded the previous reports that the protein coat of the virus remained outside the bacterial cell (as we know now, see fig. 4.2).
In the next experiment, they disrupted the protein coat of unadsorbed phages by subjecting them to osmotic shock and treated the extract with DNase. They noticed that the loss of protein coat made DNA sensitive to DNase. They also observed that the DNA of phages adsorbed on the bacteria, were injected into the bacterial cell (as now we know, seen in fig 4.2). Hence they concluded that the protein has function in protection, adsorption and injection of the DNA. Eventually, they also found that the DNA not only enters the host cell but also becomes an integral part of the bacterial cell (As we now know, see fig 4.2-4.4).
(Just for info: Read the original paper by Hershey and Chase from 1952).
Later during the study, they also realized that a strong shearing force could displace the bacterial ghost (protein coat after DNA injection), for which they used a Waring blender. The ghost was observed as radiolabelled protein in upper layer or supernatant after centrifugation at low speed (fig 5.a). Hershey and Chase noticed that even after removing up to 82% of the phage protein, reproduction continued. They hence stated that the proteins had no involvement in the reproduction. Meanwhile, upto 85% of phage DNA was transferred into the bacterial cell (i.e. the radiolabelled DNA was found in the bacterial sediment, as in fig 5.b), but Hershey and Chase were unsure about its function.
In the same series of experiment, it was revealed that the protein was not passed on to the progeny but the DNA was transferred to the progeny phage
Hence, taking into consideration all the observations, they proposed that (S containing) protein had no function in phage multiplication, while (P containing) DNA may have some function. They stated that the entire T2 phage could be hence divided into genetic and non-genetic part, making up the genotype and the phenotype.
However, they still were not sure of DNA being the genetic material and expressed doubts that some proteins without sulfur may be present with the DNA. But later, when Watson and Crick discovered the structure of the DNA in 1953, DNA was accepted as the genetic material by the science community, without any other experiment.
(Just for info: Have a look at the paper by Watson and Crick, 1953 in Nature)
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Read other posts by The Biotech Notes:
Gene Mapping Using Conjugation.
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References:
Hershey and Chase (1952) Independent functions of viral protein and nuclei acid in growth of bacteriophage. The Journal of General Physiology 36 (1): 39-56.
Cobb (2014) Oswald Avery, DNA, and the transformation of biology. Current Biology 24 (2): R55-R60.
Avery et al. (1944) Studies on the chemical nature of the substance inducing transformation of pneumococcal types. JEM. 79 (2): 137.
Mansour (2017) Bacteriophages are natural gift, could we pay further attention!. Journal of Food Microbiology. 1. 22.
Alnaji (2018) Lecture 2 The Hereditary Molecule.
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