BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 24 DNA: THE GENETIC ROLE, STRUCTURE, AND REPLICATION

24.2. Pneumococcal Transformation by DNA Demonstrated that Genes Consist of DNA

Pneumococcal Bacteria played a pivotal role in the Discovery of the Genetic Function of DNA.

Pneumococci are typically surrounded by a glistening, mucous polysaccharide capsule. This outer layer is essential for bacterial pathogenicity

and causes Pneumonia in humans and other susceptible mammals. Mutants lacking the polysaccharide coat are nonpathogenic. Wild-type bacteria are designated as S (from smooth) because they form smooth colonies, whereas mutant bacteria lacking a capsule are designated as R (from rough) because they form rough colonies. One group of R mutants lacks the dehydrogenase enzyme that converts UDP-glucose into UDP-glucuronate. This enzyme is required for the Synthesis of the capsular polysaccharide, which in these pneumococci consists of alternating sequences of glucose and glucuronate residues:

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In 1928, Fred Griffith discovered that a nonpathogenic R mutant could be transformed into a pathogenic S form in the following manner. He injected mice with a mixture of living R-form bacteria and heat-killed S pneumococci. Griffith's striking discovery was that this mixture proved fatal to mice, even though neither living R pneumococci nor heat-killed S pneumococci were lethal on their own. The Blood of the dead mice contained living S pneumococci. Consequently, the heat-killed S pneumococci had somehow transformed the living R pneumococci into living S pneumococci. This change was stable: the transformed pneumococci yielded pathogenic S-form offspring. It was subsequently established that such an R → S transformation could also occur in vitro. Certain Cells in a growing R-form culture were transformed into the S form upon The addition of a Cell-free extract from heat-killed S pneumococci. This discovery laid the foundation for

Fig. 24.1. Cytology/cytology/92.html">SCHEMATIC Structure OF DNA. The sugar-phosphate backbone is shown in black, and the purine and pyrimidine bases are multicolored, illustrating The Study of the Chemical Nature of the transforming principle

Fig. 24.2. Structure of a single DNA strand. Part of the chain is shown

Investigators began fractionating The Cell-free extract of heat-killed S pneumococci to determine the transforming activity of its components (Fig. 24.3). In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty published their findings. They demonstrated that "a nucleic acid of the ribose type is the fundamental active principle of the transforming extract of pneumococcus type III." The experimental evidence supporting this Conclusion was as follows: 1) elemental chemical Analysis of the purified, highly active transforming prin-

ciple yielded results close to the theoretical values for DNA; 2) the optical and electrophoretic properties, ultracentrifugal behavior, and diffusion constants of the purified material matched those of DNA; 3) extraction of Proteins or Lipids did not result in a loss of transforming activity; 4) Trypsin and Chymotrypsin had no effect on transforming activity; 5) Ribonuclease (known to hydrolyze ribonucleic acid) did not affect the transforming principle; 6) conversely, the addition of deoxyribonuclease destroyed transforming activity.

Fig. 24.3. Transformation of the nonpathogenic R pneumococcus (small colonies) and The Emergence of the pathogenic S pneumococcus (large, glistening colonies) induced by DNA from heat-killed S pneumococci

This work stands as a landmark in The history of biochemistry. Until 1944, it was widely believed that Genetic information resided in chromosomal proteins, while DNA played a secondary role. This prevailing view was decisively overturned by the discovery and rigorous proof that purified DNA possesses genetic Specificity. In 1943, Avery vividly described this research and its implications in a letter to his brother at another university (Fig. 24.4).

Fig. 24.4. From a letter written by Oswald Avery to his brother Roy in May 1943

Further confirmation of the genetic role of DNA came from studies of a virus (bacteriophage) that infects E. coli. Bacteriophage T2 consists of a core (DNA) enclosed within a protein coat. In 1951, Roger Herriott suggested that "the virus evidently acts like a tiny hypodermic syringe filled with the transforming principle; the virus as such never enters the cell; only the tail makes contact with the host cell and perhaps enzymatically drills a small hole in the outer membrane (Fig. 24.5). The nucleic acid then flows from the viral HEAD into the interior of the cell." To test this hypothesis, Alfred Hershey and Martha Chase performed the following experiment. Phage DNA was labeled with the radioactive isotope ³²P, and the protein coat with the isotope ³⁵S. These labels are highly specific because DNA contains no sulfur, whereas the protein coat contains no phosphorus. A culture of E. coli was infected with the labeled phage, which attached to the bacteria during a brief incubation period. The suspension was processed for several minutes in a Waring blender at 10,000 rpm. Under these conditions, the phage-infected cells were subjected to significant shearing forces that disrupted the connections between the Viruses and the bacteria. The suspension was then centrifuged to pellet the bacteria at the bottom of the tube. The resulting pellet contained the infected bacteria, while the supernatant fraction contained the smaller particles. By examining the ³²P and ³⁵S content in the pellet and supernatant fractions, the localization of the phage DNA and coat protein was determined. The results yielded the following data.

Fig. 24.5. Schematic diagram of bacteriophage T2 injecting its DNA into a bacterial cell

1. The majority of the phage DNA was recovered inside the bacteria.

2. Most of the phage protein was recovered in the supernatant fraction.

3. Homogenization has almost no effect on the ability of infected bacteria to produce viral progeny.

Additional experiments demonstrated that less than 1% of the ³⁵S was transferred from the parental phage particles to the phage progeny; conversely, 30% of the parental ³²P label was found in the progeny. These straightforward, compelling experiments showed that phage T2 could be physically separated into genetic and non-genetic components... The sulfur-containing protein of resting phage particles is restricted to the protective coat, which mediates the attachment of the phage particle to the bacterial cell and Functions as a device for injecting phage DNA into the cell. This protein presumably serves no function required for the intracellular growth of phage particles. The DNA, on the other hand, plays a crucial role in phage reproduction. The presented experiments do not warrant any further-reaching Conclusions regarding the chemical Nature of the phenomena observed."

The cautious tone of this conclusion should not diminish its significance. Before long, the genetic role of DNA became universally accepted. The Hershey-Chase experiments strongly corroborated the findings discovered eight years earlier by Avery, MacLeod, and McCarty using a different system. Additional evidence came from studies of DNA content in individual cells, which showed that for a given species, the DNA amount is constant across all

cells with a diploid set of Chromosomes. Haploid cells contain half as much DNA.