Classic experiments: DNA as the genetic material
Experiments by Frederick Griffith, Oswald Avery and his colleagues, and Alfred Hershey and Martha Chase.
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 composition.
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.
- Rough strain (nonpathogenic). When this strain is injected into a mouse, the mouse lives.
- Smooth strain (pathogenic). When this strain is injected into a mouse, the mouse gets pneumonia and dies.
- Heat-killed smooth strain. When heat-killed smooth cells are injected into a mouse, the mouse lives.
- 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.
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 identity.
Several lines of evidence suggested to Avery and his colleagues that the transforming principle might be DNA:
- 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 principle.
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 protein.
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 , 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 , 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.
- 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.
- Bacteria were infected with the phage.
- The cultures were blended and centrifuged to separate the phage from the bacteria.
- 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)
When Hershey and Chase measured radioactivity in the pellet and supernatant from both of their experiments, they found that a large amount of appeared in the pellet, whereas almost all of the 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.
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.
Want to join the conversation?
- it was good
but i wanted to ask that why do twins have different dna structure??(12 votes)
- It happens, because normally there is only one egg at the time of ovulation in the ovarian duct. And if a sperm is present there is more than enough of spermatozoids. So one lucky sperm fertilizes one egg. This way an zygote is produced which later becomes embryo and eventually a baby.
But in some cases, there is two or even more eggs in the ovarian duct, and as we know there are hundreds of sperms, both can get fertilized. In this case two different zygotes form, hence the name "dizygotic" twins. In more simple way, it is like getting pregnant twice, when female body gives two eggs that can be fertilized. And different eggs have different sets of DNA (because of myosis, crossover and other things). So dizygotic twins are related as much as simple brothers (or sisters).(17 votes)
- I don't understand how the mice died with the smooth strain but not the heat killed smooth strain.(5 votes)
- Because the heat killed smooth strain have been killed and so those dead bacteria can no longer grow and reproduce in numbers and overwhelm the mouse and kill it.(18 votes)
I'm kind of confused on Avery's experiment on transforming factors. I understand the process of it, but I don't know what exactly was grown in the petri dish, or how it proved that DNA is the transforming molecule.
Thank you,(9 votes)
- There were three major components to their experiment: (1) separated, subcellular components of heat-kill virulent S. pneumoniae (2) non-virulent S. pneumoniae and (3) mice. To see which subcellular component (the "purified substance," as Khan refers to it) was the transforming principle (responsible for "passing on" virulence to non-virulent bacteria as shown in Griffith's experiment), they systematically injected one type of substance per trial into non-virulent bacteria. This initially non-virulent bacteria then was injected into mice; if the mice lived, then the substance injected could not have been the transforming principle. They then noticed that one specific substance (which we now know is DNA) transformed the initially non-virulent bacteria and killed the mice when injected.
The results of three other tests then gave these scientists additional evidence that supported DNA as the transforming principle, which are listed in the article. The first two should be self explanatory: the substance tested positive for DNA detection and negative for protein detection. Its chemical composition also matched that of DNA. The last one may be a little confusing without knowing about the experimental setup above. They essentially treated the substance with either (1) protein/RNA degrading enzymes or (2) DNA degrading enzymes. Injecting the substance treated with (1) showed that non-virulent bacteria were still transformed (became virulent) and mice still died. Therefore, the transforming principle could not be protein/RNA, which had been degraded by enzymes in that sample. Injecting the substance treated with (2) showed that non-virulent bacteria were not transformed and mice did not die. This suggested that DNA was indeed the transforming principle because when it was degraded, virulent bacteria were unable to "pass on" their virulence.(9 votes)
- You know how DNA makes us different from any other person, well could it be so complex that we could have an attached mutation that makes us different from the human race?(4 votes)
- Mutations are present in every individual. It is part of life, the older we get the more mutations we acquire. Mutations occur in our DNA, its a structural change (a mutation is not necessarily attached to our chromosome) that alters our DNA, these alterations can be large or small. So you can think of a mutation as a genetic change in the sequence of our DNA. For example, individuals with the disease Sickle Cell Anemia are still human (and part of the human race), but they have a mutation in their genome.
So to answer your question: mutations do not necessarily make us different from the human race-because you would still have the same human genetic makeup as everyone else. The only difference between you and other people is that your DNA is slightly altered (with a mutation).(7 votes)
- What does the word transformation mean?(4 votes)
- Transformation in normal, everyday language, means to change something.
In the bacterial transformation experiment, Griffith saw that the R strain of bacteria could be changed, or 'transformed' into S-strain bacteria when they were mixed with dead S-strain bacteria.
Nowadays, 'transformation' has a very specific meaning in biology. It describes the process of bacteria taking up DNA directly from their surroundings, like in Griffith's experiment. There are also other ways DNA can get into bacteria, and they have their own names. 'Transduction' is when DNA is injected into bacteria by phage (like mentioned in Hershey and Chase's experiment above), and under certain circumstances, DNA can also be passed from one bacterium to another by 'conjugation'.
Also, another separate meaning of transformation in biology is when normal cells become 'transformed' to cancer cells.(4 votes)
- what evidence was there to lead hershey and chase to conclude that only DNA from the bacteriophage entered the bacterium leaving protein sheath behind(4 votes)
- Hershey and Chase attached atom tags to DNA and proteins from the bacteriophage. I believe it was sulfur and phosphorus. Then, they looked in the bacterium and saw only the tag they attached to the DNA, sulfur I think.(2 votes)
- I still do not fully understand how the rough strain bacteria kill the mice when there's heat killed smooth strain bacteria. Do they incorporate the dead bacteria's DNA into theirs and start replicating it and producing proteins based on that newly acquired sequence? how does that happen or why? Is that similar to what viruses do?(2 votes)
- When bacteria incorporate DNA outside of themselves into their genome, it's called transformation. This is what happened in the experiment, as the rough bacteria sucked up the still-intact DNA from the dead smooth bacteria. Yes, they did incorporate the dead bacteria's DNA into theirs and started replicating it to produce proteins from the newly acquired sequence. By the way, what viruses do is slightly different--they inject their own DNA into the bacteria which is called transduction.(4 votes)
- How did Hershey found out the precise location of the radioactive source at the end? Is it like if you take your Geiger counter near the source, the count rate increases?(2 votes)
- Probably something like that. Or you tip the supernatant to one place, and keep the pellet in your tube, and then you can detect them seperately. A geiger counter is one way, another way to detect radioactivity is using photographic film.
Edit: They did various experiments with the pellets, but in the end it looks like they dried their samples and used a geiger counter to measure how radioactive each fraction was(2 votes)
- What is the function of the enzymes protease, RNase, and DNase in relation to the mice experimentation?(2 votes)
- RNase and DNases are not designed/used or connected specifically to the mice.
Mice are used as model organisms, but it means that the same DNase would have the same effect on Caenorhabditis Elegans (nematode model organism).
RNase is a type of enzymes that cut RNA molecules, and DNase is enzymes (endonucleases or exonucleases) that specifically cut DNA molecules. But it does not matter which organism is experimented on.
Proteases on the other hand, are used to cut proteins.(2 votes)
- What made DNA able to transform R-strains into S-strains cells?(2 votes)