Chapter 1. How Do We Know 3.1

1.1 Figure 3.1

What is the nature of the genetic material?

In 1869, a German chemist, Johann Frederick Miescher, isolated a novel substance from the nuclei of white blood cells. Since it was derived from the nucleus, Miescher named the substance “nuclein." Unlike proteins, this substance had a high concentration of phosphorus along with nitrogen. Miescher’s colleagues tried to convince him that nuclein was just another protein and phosphorus was a contaminant. However, Miescher was very persistent in his belief that nuclein was a new type of molecule. Eventually, chemical analysis revealed that chromosomes are composed of both protein and Miescher’s nuclein, which we now call deoxyribonucleic acid (DNA).

Of these two substances, nucliec acids and protein, which is the genetic material? In the first half the 20^th century, this question was vigorously debated. Almost everyone thought that proteins must be the genetic material, for they knew that the molecule of hereditary had to be able to contain an extraordinary amount of information. It did not seem possible that a simple molecule like DNA could be the genetic material.

This set the stage for the British physician Frederick Griffith in 1928. Griffith was an army medical officer and was attemptng to develop a vaccine against Streptococcus pneumoniae, a bacterium that is one cause of the lung disease pneumonia. Griffith hoped that either heat-killed virulent strains (denoted “S” since they form smooth colonies as a result of a polysaccharide capsule) or live nonvirulent strains (denoted “R” because they form rough colonies and lack the capsule) could be used as a vaccine. As seen in the text, Griffith never did develop a vaccine, but his work opened a door to the molecular world of heredity. This is not unusual in science, where research in one area unexpectedly sheds light on another area.

Part 1

Instructions: Read through Figure 3.1 carefully and then answer the questions below:

In designing experiments, researchers often set up controlled experiments. In a controlled experiment, several groups are tested simultaneously, keeping as many variables the same among them. In one group, a single variable is changed, allowing the researcher to see if that variable has an effect on the results of the experiment. This is known as the test group. In another group, the variable is not changed and no effect is expected. This is known as a negative control. Finally, in a third group, a variable is deliberately introduced that has a known effect, to be sure that the experiment is working properly. This is known as a positive control.

For example, if you are interested in whether a new medicine is effective in the treatment of headaches, you may have three groups of patients - one group might receive no medicine (the negative control), one group might receive the medicine (the test group), and one group might receive a medicine known to be effective against headaches (the positive control). All of the other variables, such as age, gender, socioeconomic backgroup, and the like, would be similar among the three groups. In some cases, researchers even control for the act of giving a medicine. In this case, the negative control might be a placebo, a sugar pill with no pharmacologic effect. In this way, all three groups receive medicine, so the researchers have controlled even for this potential variable.

When performing experiments, researchers manipulate the test group differently than the control groups.The difference is known as a variable, and variables are of two types. An independent variable is the manipulation performed on the test group. The dependent variable is the effect in the test group after the manipulation has been applied.

Part 2

Hammerling first decided to perform a series of general experiments as a foundation. He removed the cap of one alga then allowed it to grow. The organism grew back the cap in the original shape. He removed the stalk and cap of another alga then allowed it to grow. The organism grew back the stalk and cap in the original shape. He removed the base of a last alga and then allowed it to grow. The alga died.

With these initial tests, Hammerling proposed that something in the base seemed to be controlling cell function and the development of traits. He concluded that it must be something in the conspicuous little ball in the base known as the nucleus. With the knowledge from his previous experiments, Hammerling decided to perform a hybridization. This invloved taking two similar species of Acetabularia (mediterranea and crenulata) and grafting them together to see if the traits would alter in the resultant hybrid. The initial, major anatomical difference between the two organisms was the shape of the cap, as depicted below:

Hammerling thought to use his intial organism, A. mediterranea, as the base to which he would supplement a portion of the new organism. From A. mediterranea he removed the cap and stalk, leaving only the intact base. From A. crenulata, he removed the cap and base, leaving only the intact stalk. He then grafted the new stalk onto the the original base.

To continue this line of questioning about the nature of genetic material and its connection with the cell’s nucleus, we need to explore other contributions, such as those by Thomas King and Robert Briggs. Before Briggs and King performed their landmark work, Yves Delage in 1895 first wrote about a theoretical process known as nuclear transplantation (which currently is better known as cloning: making an exact genetic copy of a cell or organism). Delage hypothesized that if the nucleus of an egg were replaced with that of another, full development might occur. However, Delage did not have the technology available to him to perform these experiments. In 1938, Hans Spemann envisioned a “fantastical cloning experiment” where one could remove the nucleus of an egg and replace it with a different nucleus taken from a developed cell. Nearly fifteen years after Spemann, Thomas King and Robert Briggs utilized Rana pipiens, northern leopard frogs as a model depicted below.

Briggs intended to transfer the nucleus of a blastula cell into a fertilized egg whose nucleus was removed. A glass pipette wider than the cell’s nucleus but smaller than the cell width was inserted. This allowed the cell to be crushed, leaving only the nucleus intact inside the pipette. The nucleus of the blastula cell was then inserted into the enucleated egg through an incision cut into the egg’s coating.

Briggs’ and Kings’ early attempts failed, but the time the project was completed, they had successfully created 35 complete embryos and 27 tadpoles from 104 successful nuclear transplants. As an extension of this work Briggs, King, and later John Gurdon attempted to transfer nuclei of older embryonic cells and even differentiated cells of tadpoles. Differentiated implies that these cells have received cell fate and are developing into specific tissues.

Summary

As seen earlier with Hammerling’s experiments, the resulting hybrid cell had portions from both species of Acetabularia, and the hybrid grew a cap structure that had morphologies resembling a mixture of the cap anatomies. However, when the hybrid cell divided, the cap morphology went back to the original shape of A. mediterranea, which was the donor base (containing an intact nucleus) for the hybrid. With this, Hammerling was able to determine that the nucleus of a cell controls the development of organisms by containing the hereditary information.

As seen with the work of Briggs and King, the nucleus again demonstrated its power by controlling the embryological development of frogs.

Upon the shoulders of scientists like Hammerling, Levene, Meselson, Stahl, Sanger, Watson, Crick, Franklin, Avery, Wilkins, Chargaff, Briggs, King, MacLeod, McCarty, and Griffith, a new realm of science was emerging … that of molecular biology and its application, biotechnology. Biotechnology is any technique that utilizes living organisms or substances from those oraganisms, to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses. This exciting and diverse field touches the medical and phramaceutical fields, environmental sciences, agriculture, bioremediation, forensics, and synthesis of industrial chemicals/processes. It is also has given us great insight and further validation of the role and power of nucleic acids.

Part 3

So, how can aspects of this scientific field exemplify the nature of genetic material? We shall explore this question next and see that it is an extension of the seeds that inidividuals like Hammerling already had planted.

Prokaryotes are types of primitive cells that lack organelles (specialized membrane-bound structures which perform specific functions for the cell), such as bacteria. Because they lack organelles, their DNA is not enclosed in any structure and is therefore spread out into the cell in a large circular molecule (chromosomal), attaching to the cell’s main external barrier (cell membrane) here and there. This large molecule contains those instructions necessary for daily functioning; however, smaller, semi-circular pieces of DNA exist outside of it. These are called plasmids and usually contain non-essential life instructions (please see below).

Plasmid DNA can be taken in by a new bacterial cell or artificially manipulated and given to a bacterial cell, thereby denoting new capabilities and traits the cell did not have prior. In other words, bacteria have the ability to take on stray pieces of DNA from dead cells and its surrounding environment to potentially improve its chances for survival. This is a transformation, as already cited in the text. For example, ampicillin is an antibiotic frequently used to treat bacterial infections, as it will interfere with the cell’s ability to successfully divide. The following is an exercise to exemplify this ability.

Now let’s introduce a plasmid into the environment as depicted below. This plasmid has a shaded region indicated as “amp”. This means that this circular piece of DNA has a DNA sequence which would allow for resistance to ampicillin, an antibiotic. This plasmid is now transformed into an E. coli cell.

What do think will happen if it this transformed cell is placed onto a sterile growth media plate without the ampicillin antibiotic and another with the ampicillin antibiotic?