While chemists were working out the structure of DNA, biologists were attempting to identify the source of genetic information. Mendel identified the basic rules of heredity in 1866, but he had no idea about the physical nature of hereditary information. By the early 1900s, biologists had concluded that genes reside on chromosomes, which were known to contain both DNA and protein. Two sets of experiments, one conducted on bacteria and the other on viruses, provided pivotal evidence that DNA, rather than protein, was the genetic material.

THE DISCOVERY OF THE TRANSFORMING PRINCIPLE The first clue that DNA was the carrier of hereditary information came with the demonstration that DNA was responsible for a phenomenon called transformation (see Chapter 7). This phenomenon was first observed in 1928 by Fred Griffith, an English physician whose special interest was the bacterium that causes pneumonia, Streptococcus pneumoniae. Griffith had succeeded in isolating several different strains of S. pneumoniae (type I, II, III, and so forth). In the virulent (disease-causing) forms of a strain, each bacterium is surrounded by a polysaccharide coat, which makes the bacterial colony appear smooth (S) when grown on an agar plate. Griffith found that these virulent forms occasionally mutated to nonvirulent forms, which lack a polysaccharide coat and produce a rough-appearing colony (R).

Griffith observed that small amounts of living type IIIS bacteria injected into mice caused the mice to develop pneumonia and die; when he examined the dead mice, he found large amounts of type IIIS bacteria in their blood (Figure 8.1a). When Griffith injected type IIR bacteria into mice, the mice lived, and no bacteria were recovered from their blood (Figure 8.1b). Griffith knew that boiling killed all bacteria and destroyed their virulence; when he injected large amounts of heat-killed type IIIS bacteria into mice, the mice lived, and no type IIIS bacteria were recovered from their blood (Figure 8.1c).

The results of these experiments were not unusual. However, Griffith got a surprise when he injected his mice with a small amount of living type IIR bacteria along with a large amount of heat-killed type IIIS bacteria. Because both the type IIR bacteria and the heat-killed type IIIS bacteria were nonvirulent, he expected these mice to live. Surprisingly, 5 days after the injections, the mice became infected with pneumonia and died (Figure 8.1d). When Griffith examined blood from the hearts of these mice, he observed live type IIIS bacteria. Furthermore, these bacteria retained their type IIIS characteristics through several generations: the infectivity was heritable.

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Griffith concluded that the type IIR bacteria had somehow been transformed, acquiring the genetic virulence of the dead type IIIS bacteria, and that this transformation had produced a permanent genetic change in the bacteria. Although Griffith didn’t understand the nature of transformation, he theorized that some substance in the polysaccharide coat of the dead bacteria might be responsible. He called this substance the transforming principle. TRY PROBLEM 19

IDENTIFICATION OF THE TRANSFORMING PRINCIPLE At the time of Griffith’s report, Oswald Avery was a microbiologist at the Rockefeller Institute. At first, Avery was skeptical of Griffith’s results, but after other microbiologists successfully repeated Griffith’s experiments with other bacteria, Avery set out to understand the nature of the transforming principle.

After 10 years of research, Avery, Colin MacLeod, and Maclyn McCarty succeeded in isolating and partially purifying the transforming substance. They showed that it had a chemical composition closely matching that of DNA and quite different from that of proteins. Enzymes such as trypsin and chymotrypsin, known to break down proteins, had no effect on the transforming substance. Ribonuclease, an enzyme that destroys RNA, also had no effect. Enzymes capable of destroying DNA, however, eliminated the biological activity of the transforming substance (Figure 8.2). Avery, MacLeod, and McCarty showed that the transforming substance precipitated at about the same rate as purified DNA and that it absorbed ultraviolet light at the same wavelengths as DNA. These results, published in 1944, provided compelling evidence that the transforming principle—and therefore genetic information—resides in DNA. However, new theories in science are rarely accepted on the basis of a single experiment, and many biologists continued to prefer the hypothesis that the genetic material is protein.

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THE HERSHEY–CHASE EXPERIMENT A second piece of evidence that indicated DNA was the genetic material resulted from a study of the T2 bacteriophage conducted by Alfred Hershey and Martha Chase. The T2 bacteriophage infects the bacterium Escherichia coli (Figure 8.3a). As we saw in Chapter 7, a phage reproduces by attaching to the outer wall of a bacterial cell and injecting its DNA into the cell, where it replicates and directs the cell to synthesize phage protein. The phage DNA becomes encapsulated within the proteins, producing progeny phages that lyse (break open) the cell and escape (Figure 8.3b).

At the time of the Hershey–Chase study (their paper was published in 1952), biologists did not understand exactly how phages reproduce. What they did know was that the T2 phage is approximately 50% protein and 50% DNA, that a phage infects a cell by first attaching to the cell wall, and that progeny phages are ultimately produced within the cell. Because the progeny carry the same traits as the infecting phage, they knew that genetic material from the infecting phage must be transmitted to the progeny, but how this transmission takes place was unknown.

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Hershey and Chase designed a series of experiments to determine whether the phage protein or the phage DNA is transmitted in phage reproduction. To follow the fates of protein and DNA, they used radioactive forms, or isotopes, of phosphorus and sulfur. A radioactive isotope can be used as a tracer to identify the location of a specific molecule because any molecule containing the isotope will be radioactive and therefore easily detected. DNA contains phosphorus, but not sulfur. Thus, Hershey and Chase used a radioactive isotope of phosphorus (³²P) to follow phage DNA during reproduction. Protein contains sulfur, but not phosphorus, so they used a radioactive isotope of sulfur (³⁵S) to follow the protein.

Hershey and Chase grew one batch of E. coli in a medium containing ³²P and infected the bacteria with T2 phage so that all the new phages would have DNA labeled with ³²P (Figure 8.4). They grew a second batch of E. coli in a medium containing ³⁵S and infected these bacteria with T2 phage so that all these new phages would have proteins labeled with ³⁵S. Hershey and Chase then infected separate batches of unlabeled E. coli with the ³⁵S- and ³²P-labeled phages. After allowing time for the phages to infect the cells, they placed the E. coli cells in a blender and sheared off the now-empty phage protein coats from the cell walls. They separated out the protein coats and cultured the infected bacterial cells.

When phages labeled with ³⁵S infected the bacteria, most of the radioactivity was detected in the protein coats and little was detected in the cells. Furthermore, when new phages emerged from the cells, they contained almost no ³⁵S (see Figure 8.4). This result indicated that the protein component of a phage does not enter the cell and is not transmitted to progeny phages.

In contrast, when Hershey and Chase infected bacteria with ³²P-labeled phages and removed the protein coats, the bacteria were radioactive. Most significantly, after the cells lysed and new progeny phages emerged, many of these phages emitted radioactivity from ³²P, demonstrating that DNA from the infecting phages had been passed on to the progeny (see Figure 8.4). These results confirmed that DNA, not protein, is the genetic material of phages. TRY PROBLEM 36