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06 lab, Exams of Biochemistry

GRIFFITH/HERSHEY/CHASE: DNA IS THE GENETIC MATERIAL

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CHAPTER 6
GRIFFITH/HERSHEY/CHASE: DNA IS THE GENETIC MATERIAL
In 1928, Frederick Griffith was able to transform harmless bacteria into virulent pathogens with an extract
that Oswald Avery proved, in 1944, to be DNA. In 1952, Martha Chase and Alfred Hershey used
radioactively labeled virus DNA to infect bacteria, proving the same point. These important experiments
established that DNA is the genetic material.
IDENTIFICATION OF DNA
Deoxyribonucleic acid (DNA) was first described by Friedrich Miescher in 1869, only four years after
Mendel’s work was published. But it took over 80 years for its role as the genetic material of most
organisms to become firmly established. DNA was first characterized as acid-precipitable material from
the cell nuclei of pus and fish sperm. The proportion of nitrogen and phosphorus was very unusual
compared to other known organic substances, convincing Miescher he had discovered a new biological
substance. He called it nuclein, because it was associated exclusively with the nucleus. Further work
demonstrated that nuclein is a complex of protein and DNA.
DNA AND HEREDITY
Although clear experiments linking DNA to heredity were not performed until the mid 1940s, there was a
good deal of circumstantial evidence that this was the case. In higher organisms, DNA was found almost
exclusively in the chromosomes. The histone proteins and RNA, which chromosomes also contain, did not
seem likely candidates as genetic material; sperm contained almost no RNA, and the histones are replaced
in sperm by a different protein, protamine.
Unlike RNA and protein, every diploid cell of an organism has about the same amount of DNA. In the hen,
for example, the red blood cells contain 2.6 x 10-12g of DNA per cell, the kidney contains 2.3 x 10-12g per
cell, and the liver contains 2.6 x 10-12g per cell. Furthermore, the amount of DNA seems correlated with
chromosomal division; entering mitosis, the amount of cellular DNA doubles, while the haploid products of
meiosis have only half the normal amount (thus rooster sperm contains 1.3 x 10-12g of DNA). In polyploid
plants, which contain multiples of the diploid number of chromosomes, the quantity of DNA is also a
multiple of the diploid amount. Thus, the close association of DNA with chromosomes strongly implicates
DNA as the genetic material.
DNA CAN GENETICALLY TRANSFORM CELLS
The first unambiguous evidence that DNA was the hereditary material came from Frederick Griffith’s
studies in 1928. Griffith used chemical mutagens to isolate a nonvirulent form of the bacterium that causes
pneumonia, Diplococcus pneumoniae. Virulence required the presence of a polysaccharide capsule around
the bacterium. The nonvirulent mutants lacked this capsule. Colonies of nonvirulent capsuleless bacteria
appeared rough and were designated R. In contrast, the virulent form produced colonies that appeared
smooth, so it was designated S. Several virulent forms were known, each with a characteristic
polysaccharide capsule (called IS, IIS, IIIS, etc.), which is genetically inherited and is immunologically
distinct from other forms.
A smooth bacterium of a particular capsule type (say IIS) can mutate to a nonencapsulated, nonvirulent
form (IIR, because it derives from a type II cell). This happens at a very low frequency (in less than one in
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CHAPTER 6

GRIFFITH/HERSHEY/CHASE: DNA IS THE GENETIC MATERIAL

In 1928, Frederick Griffith was able to transform harmless bacteria into virulent pathogens with an extract that Oswald Avery proved, in 1944, to be DNA. In 1952, Martha Chase and Alfred Hershey used radioactively labeled virus DNA to infect bacteria, proving the same point. These important experiments established that DNA is the genetic material.

IDENTIFICATION OF DNA

Deoxyribonucleic acid (DNA) was first described by Friedrich Miescher in 1869, only four years after Mendel’s work was published. But it took over 80 years for its role as the genetic material of most organisms to become firmly established. DNA was first characterized as acid-precipitable material from the cell nuclei of pus and fish sperm. The proportion of nitrogen and phosphorus was very unusual compared to other known organic substances, convincing Miescher he had discovered a new biological substance. He called it nuclein , because it was associated exclusively with the nucleus. Further work demonstrated that nuclein is a complex of protein and DNA.

DNA AND HEREDITY

Although clear experiments linking DNA to heredity were not performed until the mid 1940s, there was a good deal of circumstantial evidence that this was the case. In higher organisms, DNA was found almost exclusively in the chromosomes. The histone proteins and RNA, which chromosomes also contain, did not seem likely candidates as genetic material; sperm contained almost no RNA, and the histones are replaced in sperm by a different protein, protamine.

Unlike RNA and protein, every diploid cell of an organism has about the same amount of DNA. In the hen, for example, the red blood cells contain 2.6 x 10-12g of DNA per cell, the kidney contains 2.3 x 10-12g per cell, and the liver contains 2.6 x 10-12g per cell. Furthermore, the amount of DNA seems correlated with chromosomal division; entering mitosis, the amount of cellular DNA doubles, while the haploid products of meiosis have only half the normal amount (thus rooster sperm contains 1.3 x 10-12g of DNA). In polyploid plants, which contain multiples of the diploid number of chromosomes, the quantity of DNA is also a multiple of the diploid amount. Thus, the close association of DNA with chromosomes strongly implicates DNA as the genetic material.

DNA CAN GENETICALLY TRANSFORM CELLS

The first unambiguous evidence that DNA was the hereditary material came from Frederick Griffith’s studies in 1928. Griffith used chemical mutagens to isolate a nonvirulent form of the bacterium that causes pneumonia, Diplococcus pneumoniae. Virulence required the presence of a polysaccharide capsule around the bacterium. The nonvirulent mutants lacked this capsule. Colonies of nonvirulent capsuleless bacteria appeared rough and were designated R. In contrast, the virulent form produced colonies that appeared smooth, so it was designated S. Several virulent forms were known, each with a characteristic polysaccharide capsule (called I S , II S , III S , etc.), which is genetically inherited and is immunologically distinct from other forms.

A smooth bacterium of a particular capsule type (say II S ) can mutate to a nonencapsulated, nonvirulent form (II R , because it derives from a type II cell). This happens at a very low frequency (in less than one in

a million cells), but it is inherited when it does occur. Similarly, the II R cell can mutate back to the II S virulent form at low frequency. However, the II R cell line can not mutate to a III S virulent form. This property provides the key to the experiment.

Figure 6. Griffith’s discovery of the “transforming principle.”

GRIFFITH’S EXPERIMENT

Griffith mixed Pneumococcus type II R with II S cells that had been killed and rendered nonvirulent by heating them to 65°C, and he injected them into a host rabbit or, in other experiments, into a mouse. Neither strain injected alone produced disease, and no disease was expected from the mixed injections, as neither strain was virulent. However, many of the rabbits given mixed injections did come down with pneumonia and died. When analyzed, they all contained living virulent type III S cells! These cells could not have arisen from the type II R cells by mutations (they would have produced type II S cells), and the type III S cells were demonstrably dead (injected alone they caused no disease). Some factor must have passed from the dead III S cells to the live II R ones, endowing them with the ability to make a capsule of the III type. Griffith called the factor “transforming principle” and the process genetic transformation (figure 6.1).

The transforming principle could be isolated as a cell-free extract and was fully active. The stability of the principle’s transforming activity to heat treatment at 65°C suggested that it was not a protein (such high temperatures denature most proteins). In 1944, Oswald Avery, C. M. MacLeod, and M. J. McCarty succeeded in isolating a highly purified preparation of DNA from the type III S bacteria. The preparation of this type III S DNA was fully active as a transforming agent and could transform type II R cells into type III S cells in a test tube. If the DNA was destroyed by deoxyribonuclease (an enzyme that specifically attacks DNA), all transforming activity was lost. It therefore seemed clear that DNA was “functionally active in determining the biochemical activities and specific characteristics of pneumococcal cells.”

Figure 6. The Hershey-Chase experiment with bacterial viruses.

THE TOBACCO MOSAIC VIRUS (TMV)

Some viruses do not contain DNA, being made up instead of protein and RNA (ribonucleic acid). The tobacco mosaic virus (TMV) is such an RNA virus. H. Fraenkel-Conrat and others were able to dissociate the TMV into its constituent protein and RNA parts (figure 6.3). When the parts were mixed, they reformed TMV particles that were normal in every respect. That the RNA contained the genetic information was demonstrated by isolating protein and RNA from several different types of TMV, with subsequent combinations of protein and RNA mixed together. These reconstituted viruses, containing protein from one type and RNA from another, were then allowed to infect tobacco cells. In every case the progeny TMVs proved to have the protein coats of the type that had contributed the RNA, and not of the type that had contributed the protein. Thus, in the tobacco mosaic virus, the RNA, rather than the protein, must be acting as the genetic material.

Figure 6. Fraenkel-Conrat’s virus-reconstitution experiment.