In
the first half of the twentieth century, Gregor Mendel's
principles of genetic inheritance became widely accepted, but
the chemical nature of the hereditary material remained unknown. Scientists did
know that genes were located on chromosomes and that chromosomes consisted of DNA and
proteins. At the time, however, proteins seemed to be a better choice for the
genetic material, because chemical analyses had shown that proteins are more
varied than DNA in their chemical composition, as well as in their
physical properties. Therefore, the eventual identification of DNA as
the hereditary material came as a surprise to scientists. This breakthrough
resulted from a series of experiments with bacteria and
bacteriophages, or viruses that infect bacteria. Together, these
experiments demonstrated that DNA was transferred between generations
and that this molecule had the ability to transform the properties of
a cell.
Frederick
Griffith Discovers Bacterial Transformation
In
the aftermath of the deadly 1918 flu epidemic, governments across the globe
rushed to develop vaccines that could stop the spread of infectious diseases.
In England, microbiologist Frederick Griffith was studying two strains of
Streptococcus pneumoniae that varied dramatically in both their appearance and
their virulence, or their ability to cause disease. Specifically, the highly
virulent S strain had a smooth capsule, or outer coat composed of
polysaccharides, while the nonvirulent R strain had a rough appearance and
lacked a capsule (Figure 1). Mice injected with the S strain died within a few
days after injection, while mice injected with the R strain did not die.
Through
a series of experiments, Griffith established that the virulence of the S
strain was destroyed by heating the bacteria. Thus, he was surprised to find
that mice died when they were injected with a mixture of heat-killed Sbacteria
and living R bacteria, neither of which caused mice to die when they were
injected alone. Griffith was able to isolate live bacteria from the hearts of
the dead animals that had been injected with the mixed strains, and he observed
that these bacteria had the smooth capsules characteristic of the S strain.
Based on these observations, Griffith hypothesized that a chemical component
from the virulent S cells had somehow transformed the R cells into the more
virulent S form (Griffith, 1928). Unfortunately, Griffith was not able to
identify the chemical nature of this "transforming principle" beyond
the fact that it was able to survive heat treatment.
DNA Is Identified as the “Transforming Principle”
The
actual identification of DNA as the "transforming principle" was an
unexpected outcome of a series of clinical investigations of pneumococcal
infections performed over many years (Steinman & Moberg, 1994). At the same
time that Griffith was conducting his experiments, researcher Oswald Avery and
his colleagues at the Rockefeller University in New York were performing
detailed analyses of the pneumococcal cell capsule and the role of this capsule
in infections. Modern antibiotics had not yet been discovered, and Avery was
convinced that a detailed understanding of the pneumococcal cell was essential
to the effective treatment of bacterial pneumonia. Over the years, Avery's
group had accumulated considerable biochemical expertise as they established
that strains of pneumococci could be distinguished by the polysaccharides in
their capsules and that the integrity of the capsule was essential for
virulence. Thus, when Griffith's results were published, Avery and his
colleagues recognized the importance of these findings, and they decided to use
their expertise to identify the specific molecules that could transform a
nonencapsulated bacterium into an encapsulated form. In a significant departure
from Griffith's procedure, however, Avery's team employed a method for
transforming bacteria in cultures rather than in living mice, which gave them
better control of their experiments.
Avery
and his colleagues provided further confirmation for this hypothesis by
chemically isolating DNA from the cell extract and showing that it possessed
the same transforming ability as the heat-treated extract. We now consider
these experiments, which were published in 1944, as providing definitive proof
that DNA is the hereditary material. However, the team's results were not well
received at the time, most likely because popular opinion still favored protein
as the hereditary material.
Hershey and Chase Prove Protein Is Not the Hereditary Material
Protein
was finally excluded as the hereditary material following a series of
experiments published by Alfred Hershey and Martha Chase in 1952. These
experiments involved the T2 bacteriophage, a virus that infects the E. coli
bacterium. At the time, bacteriophages were widely used as experimental models
for studying genetic transmission because they reproduce rapidly and can be
easily harvested. In fact, during just one infection cycle, bacteriophages
multiply so rapidly within their host bacterial cells that they ultimately
cause the cells to burst, thus releasing large numbers of new infectious
bacteriophages. The T2 bacteriophage used by Hershey and Chase was known to
consist of both protein and DNA, but the role that each substance played in the
growth of the bacteriophage was unclear. Electron micrographs had shown that T2
bacteriophages consist of an icosahedral head, a cylindrical sheath, and a base
plate that mediates attachment to the bacterium, shown schematically in Figure
5. After infection, phage particles remain attached to the bacterium, but the
heads appear empty, forming "ghosts."
To
determine the roles that the T2 bacteriophage's DNA and protein play in
infection, Hershey and Chase decided to use radioisotopes to trace the fate of
the phage's protein and DNA by taking advantage of their chemical differences.
Proteins contain sulfur, but DNA does not. Conversely, DNA contains phosphate,
but proteins do not. Thus, when infected bacteria are grown in the presence of
radioactive forms of phosphate (32P) or sulfur (35S), radioactivity can be
selectively incorporated into either DNA or protein. Hershey and Chase employed
this method to prepare both 32P-labeled and 35S-labeled bacteriophages, which
they then used to infect bacteria. To determine which of the labeled molecules
entered the infected bacteria, they detached the phage ghosts from the infected
cells by mechanically shearing them off in an ordinary kitchen blender. The
ghosts and bacterial cells were then physically separated using a centrifuge.
The larger bacterial cells moved rapidly to the bottom of the centrifuge tube,
where they formed a pellet. The smaller, lighter phage ghosts remained in the
supernatant, where they could be collected and analyzed. During analysis,
Hershey and Chase discovered that almost all of the radioactive sulfur remained
with the ghosts, while about one-third of the radioactive phosphate entered the
bacterial cells and could later be recovered in the next generation of
bacteriophages.
From
these experiments, Hershey and Chase determined that protein formed a
protective coat around the bacteriophage that functioned in both phage
attachment to the bacterium and in the injection of phage DNA into the cell.
Interestingly, they did not conclude that DNA was the hereditary material,
pointing out that further experiments were required to establish the role that
DNA played in phage replication. In fact, Hershey and Chase circumspectly ended
their paper with the following statement: "This protein probably has no
function in the growth of intracellular phage. The DNA has some function.
Further chemical inferences should not be drawn from the experiments presented"
(Hershey & Chase, 1952). However, a mere one year later, the structure of
DNA was determined, and this allowed investigators to put together the pieces
in the question of DNA structure and function
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