Structural Biochemistry/Nucleic Acid/DNA/Avery-MacLeod-McCarty Experiment

The Avery-MacLeod-McCarty Experiment was presented by Oswald Avery, Colin MacLeod, and Maclyn McCarty in 1944. During the 1930s and early 1940s, Avery and MacLeod performed this experiment at Rockefeller Institute for Medical Research, after the departure of MacLeoirulency (measure of deadly potency). This experiment would allow them to determine if rough bacteria could be transformed into smooth bacteria, hence passing along the genetic information causing the transformation. By isolating and purifying this chemical component, they could deduce if it had characteristics of a protein or DNA molecule.

Purpose
The purpose behind this experiment was to better understand the chemical component that carries the genetic information and transforms one molecule to the next.

Procedure
Bacteria grown in petri dishes can grow spots or colonies inside the dish multiplying under certain conditions. Virulent (deadly) colonies look smooth or like tiny droplets, where as non-deadly bacteria formed rigid, uneven edges, basically rough colonies. While analyzing a certain kind of pneumonia caused by bacteria in mice, they were able to isolate a "variant" (mutant) strain that did not kill the mice. During the experiments, Avery and MacLeod injected a mouse simultaneously with "boiled" or dead smooth bacteria and live rough bacteria. Thereafter a short while they were surprised to see that the mouse died. When they took samples from the dead mice, and cultured the samples in a petri dish, Avery and MacLeod found that what grew inside the culture was in fact the smooth deadly bacteria. This suggested that something from the "dead" bacteria somehow converted the rough bacteria into smooth bacteria. The rough bacteria had been permanently converted or transformed into the smooth dangerous bacteria. They had confirmed that they could not grow smooth bacteria from the boiled culture and cause disease if the dead smooth bacteria were injected alone. This all implied that a chemical component in the smooth bacteria survived and transformed the rough bacteria into smooth. Isolating and purifying that chemical component had shown that is was DNA, NOT proteins that transferred the genetic code from the smooth to the rough.

Simpler Experimental


Here is a simpler demonstration for this experiment by Oswald Avery, Colin MacLeod, and Maclyn McCarty. There are two sets of bacteria – one is smooth (virulent), one is rough (nonvirulent).

1) They first inject deadly encapsulated bacteria into the mouse – the mouse dies at the end.

2) They then inject non-encapsulated, nonvirulent bacteria into the mouse – the mouse lives.

3) Next, they heated the virulent bacteria at a temperature that kills them and injected these bacteria into the mouse – the mouse lives.

4) After that, they then have the denatured fatal bacteria mix into the living non-encapsulated, nonfatal bacteria. The mixture was then injected into the mouse – the mouse dies.

5) Finally, they mix the live, non-encapsulated harmless bacteria with the DNA that was extracted from the heated, lethal bacteria. These “harmless” bacteria injected to the mouse after being mixed – the mouse dies.

From these experiments, Avery and his group showed that nonvirulent bacteria become deadly after mixing with the DNA of the virulent bacteria . Such a demonstration shows that nonvirulent bacteria became virulent because of the genetic information that originally came from the virulent bacteria. The protein from the virulent bacteria was already denatured during Step 3. Thus, it was DNA and not protein that transferred the genetic information to the nonvirulent bacteria.

Griffith Experiment

In 1928, Frederick Griffith performed a DNA experiment using pneumonia bacteria and mice. This experiment provided evidence that some particular chemical within cells is genetic material. The objective of the experiment was to find the material within the cells responsible for the genetic codes.

For the experiment, Griffith used Streptococcus pneumoniae, known as pneumonia. Pneumonia contains two strains - a smooth and a rough strain. The smooth strain causes pneumonia and contains a polysaccharide coating around it. The rough strain does not cause pneumonia and also lacks a polysaccharide coating. For his first experiment, Griffith took the S strain (smooth strain) and injected it into the mice. He found that the mice contracted pneumonia and ended up dying. He then took the R strain (rough strain) and injected it into the mice and found that they did not contract the pneumonia illness and survived the insertion of the strain. Through these first two experiments Griffith concluded that the polysaccharide coating on the bacteria somehow caused the pneumonia illness, so he used heat to kill the bacteria (polysaccharides are prone to heat) of the S strain and injected the dead bacteria into the mice. He found that the mice lived, which indicated that the polysaccharide coating was not what caused the disease, but rather something living inside the cell. Then he hypothesized that the heat used to kill the bacteria denatured a protein within the living cells, which caused the disease. He then injected the mice with a heat killed S strain and a live R strain, which resulted in the mice dying.

Griffith performed a necropsy on the dead mice and isolated the S strain bacteria from the corpses. He concluded that the live R strain bacteria must have absorbed the genetic material from the dead S strain bacteria, which is called transformation, a process where one strain of a bacterium absorbs genetic material from another strain of bacteria and turns into the type of bacterium whose genetic material it absorbed. Since heat denatures proteins, the protein in the bacterial chromosomes was not the genetic material. However, evidence pointed to DNA. This experiment that Griffith performed was a precursor to the Avery experiment. Avery, Macleod and McCarty followed up on the experiment because they wanted a more definitive experiment and answer.

Avery, MacLeod and McCarty used heat to kill the virulent Streptococcus pneumonia bacteria and extracted RNA, DNA, carbohydrates, lipids and proteins - which were considered possible candidates for the carriers of genetic information - from the dead cells. Each molecule was added to a culture of live non-virulent bacteria to determine which was responsible for changing them into virulent bacteria. DNA was the only molecule that turned the non-virulent cells into virulent cells, which they concluded was the genetic material within cells.