Classic experiments: DNA as the genetic material

Experiments by Frederick Griffith, Oswald Avery and his colleagues, and Alfred Hershey and Martha Chase.

Introduction

Our modern understanding of DNA's role in heredity has led to a variety of practical applications, including forensic analysis, paternity testing, and genetic screening. Thanks to these wide-ranging uses, today many people have at least a basic awareness of DNA.
It may be surprising, then, to realize that less than a century ago, even the best-educated members of the scientific community did not know that DNA was the hereditary material!
In this article, we'll look at some of the classic experiments that led to the identification of DNA as the carrier of genetic information.

Protein vs. DNA

The work of Gregor Mendel showed that traits (such as flower colors in pea plants) were not inherited directly, but rather, were specified by genes passed on from parents to offspring. The work of additional scientists around the turn of the 20th century, including Theodor Boveri, Walter Sutton, and Thomas Hunt Morgan, established that Mendel's heritable factors were most likely carried on chromosomes.
Scientists first thought that proteins, which are found in chromosomes along with DNA, would turn out to be the sought-after genetic material. Proteins were known to have diverse amino acid sequences, while DNA was thought to be a boring, repetitive polymer, due in part to an incorrect (but popular) model of its structure and composition1^1.
Today, we know that DNA is not actually repetitive and can carry large amounts of information, as discussed further in the article on discovery of DNA structure. But how did scientists first come to realize that "boring" DNA might actually be the genetic material?

Frederick Griffith: Bacterial transformation

In 1928, British bacteriologist Frederick Griffith conducted a series of experiments using Streptococcus pneumoniae bacteria and mice. Griffith wasn't trying to identify the genetic material, but rather, trying to develop a vaccine against pneumonia. In his experiments, Griffith used two related strains of bacteria, known as R and S.
  • R strain. When grown in a petri dish, the R bacteria formed colonies, or clumps of related bacteria, that had well-defined edges and a rough appearance (hence the abbreviation "R"). The R bacteria were nonvirulent, meaning that they did not cause sickness when injected into a mouse.
  • S strain. S bacteria formed colonies that were rounded and smooth (hence the abbreviation "S"). The smooth appearance was due to a polysaccharide, or sugar-based, coat produced by the bacteria. This coat protected the S bacteria from the mouse immune system, making them virulent (capable of causing disease). Mice injected with live S bacteria developed pneumonia and died.
As part of his experiments, Griffith tried injecting mice with heat-killed S bacteria (that is, S bacteria that had been heated to high temperatures, causing the cells to die). Unsurprisingly, the heat-killed S bacteria did not cause disease in mice.
The experiments took an unexpected turn, however, when harmless R bacteria were combined with harmless heat-killed S bacteria and injected into a mouse. Not only did the mouse develop pnenumonia and die, but when Griffith took a blood sample from the dead mouse, he found that it contained living S bacteria!
Diagram illustrating Frederick Griffith's experiment with S and R bacteria.
  1. Rough strain (nonpathogenic). When this strain is injected into a mouse, the mouse lives.
  2. Smooth strain (pathogenic). When this strain is injected into a mouse, the mouse gets pneumonia and dies.
  3. Heat-killed smooth strain. When heat-killed smooth cells are injected into a mouse, the mouse lives.
  4. Rough strain & heat-killed smooth strain. When these two types of cells are injected into a mouse as a mixture, the mouse gets pneumonia and dies.
_Image modified from "Griffith experiment," by Madeleine Price Ball (CC0/public domain)._
Griffith concluded that the R-strain bacteria must have taken up what he called a "transforming principle" from the heat-killed S bacteria, which allowed them to "transform" into smooth-coated bacteria and become virulent.

Avery, McCarty, and MacLeod: Identifying the transforming principle

In 1944, three Canadian and American researchers, Oswald Avery, Maclyn McCarty, and Colin MacLeod, set out to identify Griffith's "transforming principle."
To do so, they began with large cultures of heat-killed S cells and, through a long series of biochemical steps (determined by careful experimentation), progressively purified the transforming principle by washing away, separating out, or enzymatically destroying the other cellular components. By this method, they were able to obtain small amounts of highly purified transforming principle, which they could then analyze through other tests to determine its identity2^2.
Several lines of evidence suggested to Avery and his colleagues that the transforming principle might be DNA2^2:
  • The purified substance gave a negative result in chemical tests known to detect proteins, but a strongly positive result in a chemical test known to detect DNA.
  • The elemental composition of the purified transforming principle closely resembled DNA in its ratio of nitrogen and phosphorous.
  • Protein- and RNA-degrading enzymes had little effect on the transforming principle, but enzymes able to degrade DNA eliminated the transforming activity.
These results all pointed to DNA as the likely transforming principle. However, Avery was cautious in interpreting his results. He realized that it was still possible that some contaminating substance present in small amounts, not DNA, was the actual transforming principle3^3.
Because of this possibility, debate over DNA's role continued until 1952, when Alfred Hershey and Martha Chase used a different approach to conclusively identify DNA as the genetic material.

The Hershey-Chase experiments

In their now-legendary experiments, Hershey and Chase studied bacteriophage, or viruses that attack bacteria. The phages they used were simple particles composed of protein and DNA, with the outer structures made of protein and the inner core consisting of DNA.
Hershey and Chase knew that the phages attached to the surface of a host bacterial cell and injected some substance (either DNA or protein) into the host. This substance gave "instructions" that caused the host bacterium to start making lots and lots of phages—in other words, it was the phage's genetic material. Before the experiment, Hershey thought that the genetic material would prove to be protein4^4.
To establish whether the phage injected DNA or protein into host bacteria, Hershey and Chase prepared two different batches of phage. In each batch, the phage were produced in the presence of a specific radioactive element, which was incorporated into the macromolecules (DNA and protein) that made up the phage.
  • One sample was produced in the presence of 35S^{35}\text S, a radioactive isotope of sulfur. Sulfur is found in many proteins and is absent from DNA, so only phage proteins were radioactively labeled by this treatment.
  • The other sample was produced in the presence of 32P^{32}\text P, a radioactive isotope of phosphorous. Phosphorous is found in DNA and not in proteins, so only phage DNA (and not phage proteins) was radioactively labeled by this treatment.
Each batch of phage was used to infect a different culture of bacteria. After infection had taken place, each culture was whirled in a blender, removing any remaining phage and phage parts from the outside of the bacterial cells. Finally, the cultures were centrifuged, or spun at high speeds, to separate the bacteria from the phage debris.
Centrifugation causes heavier material, such as bacteria, to move to the bottom of the tube and form a lump called a pellet. Lighter material, such as the medium (broth) used to grow the cultures, along with phage and phage parts, remains near the top of the tube and forms a liquid layer called the supernatant.
  1. One batch of phage was labeled with 35S, which is incorporated into the protein coat. Another batch was labeled with 32P, which is incorporated into the DNA.
  2. Bacteria were infected with the phage.
  3. The cultures were blended and centrifuged to separate the phage from the bacteria.
  4. Radioactivity was measured in the pellet and liquid (supernatant) for each experiment. 32P was found in the pellet (inside the bacteria), while 35S was found in the supernatant (outside of the bacteria)
_Image modified from "Historical basis of modern understanding: Figure 3," by OpenStax College, Biology (CC BY 3.0)._
When Hershey and Chase measured radioactivity in the pellet and supernatant from both of their experiments, they found that a large amount of 32P^{32}\text P appeared in the pellet, whereas almost all of the 35S^{35}\text S appeared in the supernatant. Based on this and similar experiments, Hershey and Chase concluded that DNA, not protein, was injected into host cells and made up the genetic material of the phage.
Not necessarily. All living things (viruses not being considered living) have DNA as their genetic material. However, some viruses actually have ribonucleic acid, or RNA, as their genetic material. You can learn more in the section on viruses.

Remaining questions

The work of the researchers above provided strong evidence for DNA as the genetic material. However, it still wasn't clear how such a seemingly simple molecule could encode the genetic information needed to build a complex organism. Additional research by many scientists, including Erwin Chargaff, James Watson, Francis Crick, and Rosalind Franklin, led to the discovery of DNA structure, clarifying how DNA can encode large amounts of information.

Attribution:

This article is a modified derivative of "Historical basis of modern understanding," by OpenStax College, Biology, CC BY 4.0. Download the original article for free at http://cnx.org/contents/185cbf87-c72e-48f5-b51e-f14f21b5eabd@10.53.
This article is licensed under a CC BY-NC-SA 4.0 license.

Works cited:

  1. Aldridge, Susan. (2003). The DNA story. In Royal society of chemistry. Retrieved July 27, 2016 from http://www.rsc.org/chemistryworld/Issues/2003/April/story.asp.
  2. Avery, O. T., MacLeod, C. M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of Pneumococcal types. J. Exp. Med., 79(2), 137-158. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2135445/.
  3. Scarc. (2009, July 7). Oswald Avery's Pneumococcus experiments: Forerunner of the DNA story [web log post]. In The Pauling blog. Retrieved from https://paulingblog.wordpress.com/2009/07/07/oswald-averys-pneumococcus-experiments-forerunner-of-the-dna-story/.
  4. Scarc. (2009, August 18). The Hershey-Chase blender experiments [web log post]. In The Pauling blog. Retrieved from https://paulingblog.wordpress.com/2009/08/18/the-hershey-chase-blender-experiments/.

Additional references:

A gene is made of DNA. (2011). In DNA from the beginning. Retrieved from http://www.dnaftb.org/17/animation.html.
Aldridge, Susan. (2003). The DNA story. In Royal society of chemistry. Retrieved July 27, 2016 from http://www.rsc.org/chemistryworld/Issues/2003/April/story.asp.
Avery, O. T., MacLeod, C. M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of Pneumococcal types. J. Exp. Med., 79(2), 137-158. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2135445/.
Brown, T. A. (2002). The human genome. In Genomes (2nd ed.). Oxford, UK: Wiley-Liss. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK21134/.
Griffiths, A. J. F., Miller, J. H., Suzuki, D. T., et al. (2000). DNA: the genetic material. In An introduction to genetic analysis (7th ed.). New York, NY: W. H. Freeman. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22104/.
National Human Genome Research Institute. (2013, April 23). 1944: DNA is "transforming principle." In Online education kit: 1940's. Retrieved from http://www.genome.gov/25520250.
O'Connor, C. (2008). Isolating hereditary material: Frederick Griffight, Oswald Avery, Alfred Hershey, and Martha Chase. Nature Education, 1(1), 105. Retrieved from http://www.nature.com/scitable/topicpage/isolating-hereditary-material-frederick-griffith-oswald-avery-336#.
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). DNA is the genetic material. In Campbell biology (10th ed., pp. 313-315). San Francisco, CA: Pearson.
Scarc. (2009, July 7). Oswald Avery's Pneumococcus experiments: Forerunner of the DNA story [web log post]. In The Pauling blog. Retrieved from https://paulingblog.wordpress.com/2009/07/07/oswald-averys-pneumococcus-experiments-forerunner-of-the-dna-story/.
Scarc. (2009, August 18). The Hershey-Chase blender experiments [web log post]. In The Pauling blog. Retrieved from https://paulingblog.wordpress.com/2009/08/18/the-hershey-chase-blender-experiments/.
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