Question

1. Review the specific contributions of the scientists whose work led to the establishment of DNA as the genetic material: Griffith, Avery, Macleod, and McCarty, Chargaff, Hershey and Chase, Meselson and Stahl.

2. What is the meaning of genetic transformation?

3. How can you use the knowledge about the base-pairing rule (A pairs with T and C pairs with G) to calculate the % of each nucleotide in a particular genome?

4. How did Hershey and Chase establish that in phage (viruses that infect bacteria) DNA is the genetic material?

5. How did Meselson and Stahl determine whether DNA replication follows a conservative, semi--conservative, or dispersive model? You should be able to apply your understanding to predict the results using their experimental set-up if DNA replication was either conservative or dispersive.

Answer 1

1. Frederick Griffith: Bacterial transformation

In 1928, British bacteriologist Frederick Griffith conducted a series of experiments using Streptococcus pneumoniae bacteria and mice. Griffith wasn't trying to identify the genetic material, but rather, trying to develop a vaccine against pneumonia. In his experiments, Griffith used two related strains of bacteria, known as R and S.

•             R strain. When grown in a petri dish, the R bacteria formed colonies, or clumps of related bacteria, that had well-defined edges and a rough appearance (hence the abbreviation "R"). The R bacteria were nonvirulent, meaning that they did not cause sickness when injected into a mouse.

•             S strain. S bacteria formed colonies that were rounded and smooth (hence the abbreviation "S"). The smooth appearance was due to a polysaccharide, or sugar-based, coat produced by the bacteria. This coat protected the S bacteria from the mouse immune system, making them virulent (capable of causing disease). Mice injected with live S bacteria developed pneumonia and died.

As part of his experiments, Griffith tried injecting mice with heat-killed S bacteria (that is, S bacteria that had been heated to high temperatures, causing the cells to die). Unsurprisingly, the heat-killed S bacteria did not cause disease in mice.

The experiments took an unexpected turn, however, when harmless R bacteria were combined with harmless heat-killed S bacteria and injected into a mouse. Not only did the mouse develop pnenumonia and die, but when Griffith took a blood sample from the dead mouse, he found that it contained living S bacteria!

Griffith concluded that the R-strain bacteria must have taken up what he called a "transforming principle" from the heat-killed S bacteria, which allowed them to "transform" into smooth-coated bacteria and become virulent.

Avery, McCarty, and MacLeod: Identifying the transforming principle

In 1944, three Canadian and American researchers, Oswald Avery, Maclyn McCarty, and Colin MacLeod, set out to identify Griffith's "transforming principle."

To do so, they began with large cultures of heat-killed S cells and, through a long series of biochemical steps (determined by careful experimentation), progressively purified the transforming principle by washing away, separating out, or enzymatically destroying the other cellular components. By this method, they were able to obtain small amounts of highly purified transforming principle, which they could then analyze through other tests to determine its identity.

Several lines of evidence suggested to Avery and his colleagues that the transforming principle might be DNA:

•             The purified substance gave a negative result in chemical tests known to detect proteins, but a strongly positive result in a chemical test known to detect DNA.

•             The elemental composition of the purified transforming principle closely resembled DNA in its ratio of nitrogen and phosphorous.

•             Protein- and RNA-degrading enzymes had little effect on the transforming principle, but enzymes able to degrade DNA eliminated the transforming activity.

These results all pointed to DNA as the likely transforming principle. However, Avery was cautious in interpreting his results. He realized that it was still possible that some contaminating substance present in small amounts, not DNA, was the actual transforming principle^33start superscript, 3, end superscript.

Because of this possibility, debate over DNA's role continued until 1952, when Alfred Hershey and Martha Chase used a different approach to conclusively identify DNA as the genetic material.

Hershey and chase proved that DNA is a genetic material. Meselson and stahl proved that replication of DNA is semi- conservative.

2. Genetic transformation: A process by which the genetic material carried by an individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome.

Transformation of cells is a widely used and versatile tool in genetic engineering and is of critical importance in the development of molecular biology. The purpose of this technique is to introduce a foreign plasmid into bacteria, the bacteria then amplifies the plasmid, making large quantities of it.

3. By chargaff rules we can say that The amount of A always equalled the amount of T, and the amount of C always equalled the amount of G (A = T and G = C)

4. The Hershey-Chase experiments

In their now-legendary experiments, Hershey and Chase studied bacteriophage, or viruses that attack bacteria. The phages they used were simple particles composed of protein and DNA, with the outer structures made of protein and the inner core consisting of DNA.

Hershey and Chase knew that the phages attached to the surface of a host bacterial cell and injected some substance (either DNA or protein) into the host. This substance gave "instructions" that caused the host bacterium to start making lots and lots of phages—in other words, it was the phage's genetic material. Before the experiment, Hershey thought that the genetic material would prove to be protein^44start superscript, 4, end superscript.

To establish whether the phage injected DNA or protein into host bacteria, Hershey and Chase prepared two different batches of phage. In each batch, the phage were produced in the presence of a specific radioactive element, which was incorporated into the macromolecules (DNA and protein) that made up the phage.

•             One sample was produced in the presence of S35, a radioactive isotope of sulfur. Sulfur is found in many proteins and is absent from DNA, so only phage proteins were radioactively labeled by this treatment.

•             The other sample was produced in the presence of P32, a radioactive isotope of phosphorous. Phosphorous is found in DNA and not in proteins, so only phage DNA (and not phage proteins) was radioactively labeled by this treatment.

Each batch of phage was used to infect a different culture of bacteria. After infection had taken place, each culture was whirled in a blender, removing any remaining phage and phage parts from the outside of the bacterial cells. Finally, the cultures were centrifuged, or spun at high speeds, to separate the bacteria from the phage debris.

Centrifugation causes heavier material, such as bacteria, to move to the bottom of the tube and form a lump called a pellet. Lighter material, such as the medium (broth) used to grow the cultures, along with phage and phage parts, remains near the top of the tube and forms a liquid layer called the supernatant.

When Hershey and Chase measured radioactivity in the pellet and supernatant from both of their experiments, they found that a large amount of P 32, P appeared in the pellet, whereas almost all of the S35, S appeared in the supernatant. Based on this and similar experiments, Hershey and Chase concluded that DNA, not protein, was injected into host cells and made up the genetic material of the phage.

5. The Meselson-Stahl experiment

Meselson and Stahl conducted their famous experiments on DNA replication using E. coli bacteria as a model system.

They began by growing E. coli in medium, or nutrient broth, containing a "heavy" isotope of nitrogen, N15. (An isotope is just a version of an element that differs from other versions by the number of neutrons in its nucleus.) When grown on medium containing heavy N15, the bacteria took up the nitrogen and used it to synthesize new biological molecules, including DNA.

After many generations growing in the N15, the nitrogenous bases of the bacteria's DNA were all labeled with heavy N15. Then, the bacteria were switched to medium containing a "light" N14 isotope and allowed to grow for several generations. DNA made after the switch would have to be made up of N14, N, as this would have been the only nitrogen available for DNA synthesis.

Meselson and Stahl knew how often E. coli cells divided, so they were able to collect small samples in each generation and extract and purify the DNA. They then measured the density of the DNA (and, indirectly,N15 and N14) using density gradient centrifugation.

This method separates molecules such as DNA into bands by spinning them at high speeds in the presence of another molecule, such as cesium chloride, that forms a density gradient from the top to the bottom of the spinning tube. Density gradient centrifugation allows very small differences—like those between N15, N- and N14, N-labeled DNA—to be detected.

Generation 0

DNA isolated from cells at the start of the experiment (“generation 0,” just before the switch to N14 medium) produced a single band after centrifugation. This result made sense because the DNA should have contained only heavy N14, at that time.

Generation 1

DNA isolated after one generation (one round of DNA replication) also produced a single band when centrifuged. However, this band was higher, intermediate in density between the heavy N15 DNA and the light N14 DNA.

The intermediate band told Meselson and Stahl that the DNA molecules made in the first round of replication was a hybrid of light and heavy DNA. This result fit with the dispersive and semi-conservative models, but not with the conservative model.

The conservative model would have predicted two distinct bands in this generation (a band for the heavy original molecule and a band for the light, newly made molecule).

Generation 2

Information from the second generation let Meselson and Stahl determine which of the remaining models (semi-conservative or dispersive) was actually correct.

When second-generation DNA was centrifuged, it produced two bands. One was in the same position as the intermediate band from the first generation, while the second was higher (appeared to be labeled only with N14).

This result told Meselson and Stahl that the DNA was being replicated semi-conservatively. The pattern of two distinct bands—one at the position of a hybrid molecule and one at the position of a light molecule—is just what we'd expect for semi-conservative replication . In contrast, in dispersive replication, all the molecules should have bits of old and new DNA, making it impossible to get a "purely light" molecule.

Generations 3 and 4

In the semi-conservative model, each hybrid DNA molecule from the second generation would be expected to give rise to a hybrid molecule and a light molecule in the third generation, while each light DNA molecule would only yield more light molecules.

Thus, over the third and fourth generations, we'd expect the hybrid band to become progressively fainter (because it would represent a smaller fraction of the total DNA) and the light band to become progressively stronger (because it would represent a larger fraction).

The experiment done by Meselson and Stahl demonstrated that DNA replicated semi-conservatively, meaning that each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand.