8.2: The Stuff of Genes

Mendel had presented his Laws of Inheritance in 1865, but they were not widely understood—probably because they relied on a strong dose of math and statistics at a time when the quantitative biology was not in vogue. By the late 1800s we knew that all eukaryotic cells contain a nucleus. Histological staining had by then shown that nuclei contained mainly proteins and DNA. The idea that the nucleus contains genetic information was gaining traction and with the rediscovery of Mendel’s work near the end of the century came an explosion of genetic studies that led to the understanding that any organism’s observable traits are based on patterns of inheritance of paired genetic factors. We call this seminal insight Mendelian Genetics.

By 1901, Albrecht had discovered the four DNA bases (adenine, thymine, cytosine and guanine) and uracil, the substitute for thymine in RNA, earning him the 1910 Nobel Prize in Physiology or Medicine. But for many years, DNA was thought to be a small molecule made up of only its four bases. The question was: How could such a simple molecule account for the inheritance of so many different physical and physiological traits? Recall Beadle Tatum and Lederberg’s one-gene/one-enzyme hypothesis, recognizing the inheritance of enzyme activities according to Mendelian rules. Even after morphing first into the one-gene/one-protein, and then into the one-gene/one-polypeptide hypothesis, this relationship between genes and polypeptides failed to shed any light on how DNA might be the genetic material.

In fact, quite the contrary! Biochemical evidence that long polymeric DNA was the stuff of genes was slow to follow. Chains of up to twenty different amino acids, polypeptides and proteins had the potential for enough structural diversity to account for the growing number of heritable traits in a given organism. Thus, proteins seemed more likely candidates for the molecules of inheritance. The experiments you’ll read about here began around the start of World War I and lasted until just after World War II. The work of Phoebus Levene over this period revealed that DNA was no mere tetramer of deoxynucleotides, but was in fact a long polymer (check it out at Long Nucleotide Polymers. This led to very clever experiments that eventually forced the conclusion that DNA, not protein, was the genetic molecule, despite being composed of just four monomeric units. Finally, we will look at the classic work of Watson, Crick, Franklin, and Wilkins, which revealed the structure of the genetic molecule.

8.2.1. Griffith's Experiment

Fred Neufeld, a German bacteriologist studying pneumococcal bacteria in the early 1900s, discovered three immunologically different strains of Streptococcus pneumoniae (types I, II and III). The virulent strain (type III) was responsible for much of the mortality during the Spanish flu (influenza) pandemic of 1918–20. This pandemic killed between twenty million and one hundred million people, commonly because of bacterial pneumonia, a superinfection by Streptococcus pneumoniae. Viral infection may weaken the immune defense against S. pneumoniae, and a recent study suggests how: the virus causes bronchial and alveolar capillary leakage increasing susceptibility of the lungs to bacterial infection (see Influenza Causes Bronchoalveolar Capillary Leakage).

In the 1920s, Frederick Griffith was working with virulent wild-type (type III) and benign (type II) strains of S. pneumoniae. The two strains were easy to tell apart in petri dishes because the virulent strain grew as morphologically smooth (S) colonies, while the benign strain formed rough (R) colonies. As we now know, S cells are coated with a polysaccharide (mucus) capsule, which makes their colonies appear smooth. In contrast, R cell colonies look rough (i.e., don’t glisten) because they lack the polysaccharide coating. Griffith knew that injecting mice with S cells killed them within about a day! Injecting them with nonlethal R cells, on the other hand, caused no harm. He had surmised that the exposure of mice to the R strain of S. pneumoniae might immunize them against lethal infection by subsequent exposure to S cells.

To test his hypothesis, Griffith injected mice with R cells. Sometime later, he injected them with S cells. Unfortunately, the attempt to immunize the mice against S. pneumoniae was unsuccessful: both the control mice injected with S cells, and the experimental mice (that received the R-strain cells first and then S cells) all died in short order! As expected, mice injected only with R cells survived. Griffith also checked the blood of his mice for the presence of bacterial cells by growing the cells from the blood in petri dishes; he made the following observations:

• Mice injected with benign R-strain cells survived, and after he plated blood from the mice on a nutrient medium, no bacterial cells grew.
• Mice injected with S-strain cells died, and bacterial cell colonies grew from their blood.

Griffith’s experiments and results, published in 1928, are summarized in Figure 8.1.

Figure 8.1: Griffith did the experiments shown here, hoping to make antibodies against streptococcal pneumonia. Across the top are his experiments, with 2 control experiments at the left. The 2 key experiments are at the right. Neither of these yielded the desired antibodies. Instead, he discovered a chemical from dead cells that genetically transformed live bacteria (the experiment at the far right). He called the chemical a "transforming principle", what we now know as DNA.

CHALLENGE

What on the coating of S cells might make them virulent and why are R cells unable to immunize mice against virulent S cells?

Griffith then performed two other experiments, also shown in the illustration:

• He injected mice with heat-killed S cells; these mice survived. Blood from these mice contained no bacterial cells. This was expected, since heating the S cells would likely have the same effect as pasteurization has on bacteria in milk!
• Griffith also injected mice with a mixture of live R cells and heat-killed S cells, in the hope that the combination might induce immunity in the mouse, whereas injecting the R cells alone had failed. You can imagine his surprise when, far from being immunized against S cell infection, the injected mice died, and abundant S cells had accumulated in their blood.

Griffith realized that something important had happened in his experiments: in the mixture of live R cells and heat-killed S cells, something released from the dead S cells had transformed some R cells. Griffith named this “something” the transforming principle—a molecule in the debris of dead S cells that could sometimes be acquired by a few live R cells and turn them into virulent S cells.

CHALLENGE

How many of the R cells initially injected into a mouse do you think would have to acquire the transforming principle from dead S cells in order to kill the mouse so quickly? Explain your answer.

8.2.2. The Avery-MacLeod-McCarty Experiment

While Griffith didn’t know the chemical identity of his transforming principle, his experiments led to studies that proved DNA to be the stuff of genes. With improved molecular purification techniques developed in the 1930s, Oswald Avery, Colin MacLeod, and Maclyn McCarty transformed R cells in vitro (that is, without the help of a mouse!). They purified heat-killed Scell DNA, proteins, carbohydrates, and lipids and separately tested the transforming ability of each molecular component on R cells in a test tube. Since only the DNA fraction of the dead S cells could cause bacterial transformation, the three scientists concluded that DNA must be the transforming principle. Figure 8.2 summarizes their experiments. But despite the evidence, DNA was not yet readily accepted as the stuff of genes.

Figure 8.2: The experiments of Avery et al. demonstrated for the first time that the chemical “stuff of genes” (i.e., of inheritance) is DNA.

The sticking point was that DNA is composed of only the four nucleotides: monophosphates of adenosine, cytidine, guanosine, and thymidine. Scientists knew that DNA was a large polymer, but they still thought of it as a simple molecule—a polymer made up of repeating sequences of the four bases (e.g., -A-G-C-T-A-G-C-T-A-G-C-T-A-G-C-T-A-G-C-T…).

It seemed that only the endless possible combinations of the twenty amino acids in proteins seemed to promise the biological specificity necessary to account for an organism’s many genetic traits. Lacking structural diversity, DNA was explained as a mere scaffold for protein genes.

To adapt Marshall McLuhan’s famous statement: “The medium is the message” (i.e., airwaves do not merely convey but are the message), many scientists still believed that proteins were the medium of genetic information as well as the functional message itself. The reluctance of influential scientists of the day to accept DNA as a transforming principle deprived its discoverers of the Nobel Prize stature they deserved. After new evidence made further resistance to that idea untenable, even the Nobel Committee admitted its error in failing to award a Nobel Prize for the discoveries of Avery, MacLeod, and McCarty. It took the experiments of Alfred Hershey and Martha Chase finally put to rest any notion that proteins were genes.

167-2 Transformation In and Out of Mice - Griffith, McCarty, et. al.

8.2.3. The Hershey-Chase Experiment

Earlier we learned that viruses cannot live an independent life! A bacterial virus, called a bacteriophage (or just phage for short), consists of DNA inside a protein capsule. The life cycle of bacterial viruses is shown in Figure 8.3.

Figure 8.3: Life cycle of a bacteriophage: the phage’s coat remains attached to a cell after infection, but the chemicals inside the phage enter the infected cell.

Phage are inert particles until they bind to and infect bacterial cells. Phage added to a bacterial culture can be seen attached to bacterial surfaces with an electron microscope. Investigators found that they could detach phage particles from bacteria simply by agitation in a blender (like one you might have in your kitchen). Centrifugation then separated the bacterial cells in a pellet at the bottom of the centrifuge tube, leaving the detached phage particles in the supernatant.

By adding phage to bacteria, then detaching them from the bacteria at different times, investigators were able to determine how long the phage had to remain attached before the bacteria would become infected. It turned out that pelleted cells that had been attached to phage for short times would survive and reproduce when resuspended in a growth medium, but pelleted cells left attached to phage for longer times would become infected. When centrifugally separated from the detached phage particles and resuspended in fresh medium, these infected cells would go on to lyse, releasing new phage. Therefore, the transfer of genetic information for virulence (i.e., infectivity) from the phage to cells took some time. Furthermore, the viral genetic material responsible for infection was apparently no longer associated with the phage capsule, which could be recovered from the centrifugal supernatant.

Alfred Hershey and Martha Chase designed an experiment to determine whether the DNA that was enclosed by the viral protein capsule or the capsule protein itself caused a phage to infect the bacterium. In the experiment, they separately grew Escherichia coli cells infected with T2 bacteriophage in the presence of either \({}^{32} \rm P\) or \({}^{35} \rm S\) (radioactive isotopes of phosphorous and sulfur, respectively). The result was to generate phage that contained either radioactive DNA or radioactive proteins, but not both (recall that only DNA contains phosphorous and only proteins contain sulfur). They then separately infected fresh E. coli cells with either \({}^{32} \rm P-\) or \({}^{35} \rm S-\) labeled radioactive phage. Their experiment is illustrated in Figure 8.4.

Figure 8.4: Alfred Hershey and Martha Chase demonstrated that the chemical stuff of viral genes is DNA.

Phage and cells were incubated with either \({}^{32} \rm P\) or \({}^{35} \rm S\) just long enough to allow infection. Some of each culture was allowed to go on and lyse to prove that the cells were infected. The remainder of each mixture was sent to the blender. After centrifugation of each blend, the pellets and supernatants were examined to see where the radioactive proteins or DNA had gone. From the results, the \({}^{32} \rm P\) always ended up in the pellet of bacterial cells, while the \({}^{35} \rm S\) was found in the phage remnant in the supernatant.

Hershey and Chase concluded that the genetic material of bacterial viruses was DNA, not protein, and that—just as Avery’s group had suggested—DNA was the bacterial transforming principle. Given the earlier resistance to “simple” DNA being the genetic material, Hershey and Chase used cautious language in framing their conclusions. They need not have; all subsequent experiments confirmed that DNA was the genetic material. Concurrent with these confirmations were experiments demonstrating that DNA might not be (indeed, was not) such a simple, uncomplicated molecule! For their final contributions to pinning down DNA as the “stuff of genes,” Hershey shared the 1969 Nobel Prize in Physiology or Medicine with Max Delbrück and Salvador E. Luria. So why didn’t Martha Chase get a share of the recognition in the Nobel Prize, given that she was Hershey’s sole coauthor on the paper presenting their findings? You may well ask!

168 Hershey and Chase: Viral Genes Are in Viral DNA

In a later chapter you will read about our benevolent (even crucial) microbiomes and the phenomenon of horizontal gene transfer (essentially, what Griffith discovered). Under the rubric not only in the lab, you can read about the role of horizontal gene transfer how gut bacteria can become harmful in A link to the full paper is in Nature Communications at Not Only In the Lab: Gut Bacteria Breaking Bad.