Chapter 1. How Do We Know 3.1

Background Information

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).

Hämmerling’s Experiment

Now that you’ve seen how Griffiths answered the question of what the genetic material is, let’s explore another experiment that tried to answer the same question with a different organism.

When nucleic acids were discovered and chemically characterized in the early 1900’s, scientists were unsure about the molecule’s function. Two major groups developed in which one favored the hypothesis that proteins carry genetic information, and the other, smaller group sided with nucleic acids. To settle this debate, key experiments were required. As already discussed, Frederick Griffith’s experiments in 1928 indicated that genetic information can be transferred between bacterial cells, but did not identify the molecule itself. However, follow-up experiments by researchers like Oswald Avery, Colin MacLeod, Maclyn McCarty, and others identified DNA as the genetic material.

Another key follow-up experiment was performed by a Danish-German scientist named Joachim Hämmerling. In the 1930’s and 1940’s, he did experiments using a marine alga (Acetabularia mediterranea) as his model organism. The alga has a very simple anatomy including a cap, stalk, and base, as shown below. The base houses a structure known as the nucleus.

Hämmerling decided to see what would happen if he removed different parts of the alga. He first removed the cap of one alga and allowed it to grow. The organism grew back the cap in the original shape. He then removed the stalk and cap of another alga and allowed it to grow. The organism grew back the stalk and cap in the original shape. Finally, he removed the base of a third alga and allowed the stalk and cap to grow. The baseless alga died.

With these initial tests, Hämmerling proposed that something in the base seemed to be controlling cell function and the development of traits. He hypothesized that it must be something in the conspicuous little ball in the base known as the nucleus. With the knowledge from his previous experiments, Hämmerling decided to cut and join (graft) structures from two similar species of Acetabularia (A. mediterranea and A. crenulata). These species are very similar, except that A. mediterranea has a round cap and A. crenulata has a star-shaped cap, as shown below:

Hämmerling used his initial organism, A. mediterranea, as the base to which he would graft a portion of the new organism, A. crenulata. 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 A. crenulata stalk onto the A. mediterranea base.

Hämmerling's Experiment

Hämmerling then watched to see what happened. He found that his hybrid alga first grew back a cap intermediate in shape between A. mediterranea and A. crenulata. When he removed the cap and allowed the alga to grow again, this time it grew back a round cap typical of A. mediterranea.

Briggs and King’s Experiment

To continue this line of questioning about the nature of the 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 is a current method of cloning, or 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 cell, development might still occur, guided by the genetic information in the transplanted nucleus. However, Delage did not have the technology to perform these experiments. Thomas King and Robert Briggs first tried this type of experiment, using the northern leopard frog, Rana pipiens, as a model organism.

King and Briggs transferred the nucleus of a blastula cell into a fertilized egg whose nucleus was removed. To do so, they inserted a glass pipette into the cell of a frog blastula, an early embryonic stage of development, and removed the cell’s nucleus. They then inserted the nucleus into an egg cell whose own nucleus had been removed (enucleated). The egg with its new nucleus is then allowed to go through normal development.

What is the major independent variable in the experiment just described?

Briggs and King's early attempts failed. However, their later attempts were successful. By the time the experiment was completed, they had successfully obtained 35 embryos and 27 tadpoles from 104 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 means that these cells are restricted in the kinds of cells they can become and are developing into specific tissues. Here is an example of the kind of data these researchers obtained:

DIY Experiment

Let's now try an experiment that will give us more information about the nature of the genetic material.

Prokaryotes are types of cells that lack organelles (specialized membrane-bound structures that perform specific functions for the cell), such as bacteria. Because they lack organelles, their DNA is not enclosed in any structure. This genomic DNA contains instructions necessary for daily function. Prokaryotes sometimes have small circular pieces of DNA in addition to their genomic DNA. These are called plasmids.

Plasmid DNA can be released and then taken up by a new bacterial cell, or artificially manipulated and introduced into a bacterial cell in the lab, thereby giving the recipient bacteria new capabilities and traits the cell did not originally have. For example, genes that confer antibiotic resistance are often carried on plasmids. The process by which a cell takes up foreign DNA is known as transformation.

Question 1.15

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{"title":"No Growth","description":"Try again. If supplied with favorable growth conditions, would you expect bacteria to grow?","type":"incorrect","color":"#99CCFF","code":"[{\"shape\":\"rect\",\"coords\":\"365,2,499,76\"}]"} {"title":"Growth","description":"Correct. This is the positive control.","type":"correct","color":"#99cc00","code":"[{\"shape\":\"rect\",\"coords\":\"361,166,499,242\"}]"}

Question 1.16

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{"title":"No Growth","description":"Correct. This is the negative control. The antibiotic kills the cells.","type":"correct","color":"#99CCFF","code":"[{\"shape\":\"rect\",\"coords\":\"365,2,499,76\"}]"} {"title":"Growth","description":"Try again. Would the presence of an antibiotic be conducive to bacterial growth, especially if the bacteria did not carry genes conferring antibiotic resistance?","type":"incorrect","color":"#99cc00","code":"[{\"shape\":\"rect\",\"coords\":\"361,166,499,242\"}]"}

Now let’s introduce a plasmid into the environment. This plasmid has a gene for resistance to the antibiotic ampicillin. This means that this plasmid has a DNA sequence which would allow for resistance to ampicillin, an antibiotic. This plasmid is now transformed into a bacterial cell.

Question 1.17

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{"title":"Growth","description":"Correct. The cells will grow with or without the addition of the plasmid carrying the ampicillin resistance gene.","type":"correct","color":"#99CCFF","code":"[{\"shape\":\"rect\",\"coords\":\"362,311,498,383\"}]"} {"title":"No Growth","description":"Incorrect. Try again.","type":"incorrect","color":"#99cc00","code":"[{\"shape\":\"rect\",\"coords\":\"365,149,498,221\"}]"}

Question 1.18

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{"title":"Growth","description":"Correct. The cells now have additional \"information\" in the form of the plasmid with the gene for ampicillin resistance, and can grow on the ampicillin plate.","type":"correct","color":"#99CCFF","code":"[{\"shape\":\"rect\",\"coords\":\"362,311,498,383\"}]"} {"title":"No Growth","description":"Try again. Transformed bacteria carry a DNA sequence that confers antibiotic resistance. Would this confer an advantage for bacteria plated on ampicillin-treated plates?","type":"incorrect","color":"#99cc00","code":"[{\"shape\":\"rect\",\"coords\":\"365,149,498,221\"}]"}

Question 1.21

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{"title":"No Growth","description":"Incorrect. Try again.","type":"incorrect","color":"#99CCFF","code":"[{\"shape\":\"rect\",\"coords\":\"362,1,497,75\"}]"} {"title":"Growth","description":"Correct. The cells will grow with or without the intact, uninterrupted \"amp\" plasmid DNA addition.","type":"correct","color":"#99cc00","code":"[{\"shape\":\"rect\",\"coords\":\"364,166,499,239\"}]"}

Question 1.22

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{"title":"No Growth","description":"Correct. Since the DNA region coding for ampicillin resistance is interrupted, the cell will lose its resistance and die when placed onto the ampicillin antibiotic plate.","type":"correct","color":"#99CCFF","code":"[{\"shape\":\"rect\",\"coords\":\"362,1,497,75\"}]"} {"title":"Growth","description":"Incorrect. Note that ScaI treatment renders the plasmid’s gene for ampicillin resistance ineffective. Would this result in antibiotic-resistant bacteria? ","type":"incorrect","color":"#99cc00","code":"[{\"shape\":\"rect\",\"coords\":\"364,166,499,239\"}]"}