In this section, you will explore the following questions:
- What is transformation of DNA? How do Griffith’s experiments in 1928 relate to our modern understanding of DNA and how it works?
- What are key historic experiments that helped identify DNA as the genetic material?
- What are Chargaff’s rules of nitrogenous base pairing?
Connection for AP® Courses
Today the three letters “DNA” have become synonymous with crime solving, paternity testing, human identification, and genetic testing. All of these procedures are possible because of the discovery, in the middle of the twentieth century, that DNA is the genetic material. The results of several classic experiments set the stage for an explosion of our knowledge about DNA and how it stores and transmits genetic information. DNA was first isolated from white blood cells by Miescher in the 1860s. Over fifty years later, Griffith’s work transforming strains of the bacterium Streptococcus pneumoniae provided the first clue that DNA and not protein (as others argued) is the universal molecule of heredity. Griffith’s conclusions were later supported by Avery, MacLeod, and McCarty.
Subsequent experiments by Hershey and Chase using the bacteriophage T2 proved decisively that DNA is the genetic material. Shortly thereafter, Chargaff determined the ratios of adenine, thymine, cytosine, and guanine in DNA, suggesting paired relationships (A = T and C = G). He also found that the percentages of A, T, C, and G are different for different species. All of these historically important experiments shaped our current understanding of DNA.
The content presented in this section supports the learning objectives outlined in Big Ideas 3 and 4 of the AP® Biology Curriculum Framework. The AP® learning objectives merge essential knowledge content with one or more of the seven science practices. These objectives provide a transparent foundation for the AP® Biology course, along with inquiry-based laboratory experiences, instructional activities, and AP® exam questions.
|Big Idea 3||Living systems store, retrieve, transmit and respond to information essential to life processes.|
|Enduring Understanding 3.A||Heritable information provides for continuity of life.|
|Essential Knowledge||3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.|
|Science Practice||6.5 The student can evaluate alternative scientific explanations.|
|Learning Objective||3.1 The student is able to construct scientific explanations that use the structures and mechanisms of DNA to support the claim that DNA is the primary source of heritable information.|
|Essential Knowledge||3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.|
|Science Practice||4.1 The student can justify the selection of the kind of data needed to answer a particular scientific question.|
|Learning Objective||3.2 The student is able to justify the selection of data from historical investigations that support the claim that DNA is the source of heritable information.|
|Big Idea 4||Biological systems interact, and these systems and their interactions possess complex properties.|
|Enduring Understanding 4.A||Interactions within biological systems lead to complex properties.|
|Essential Knowledge||4.A.1 The subcomponents of biological molecules and their sequence determine the properties of that molecule.|
|Science Practice||7.1 The student can connect phenomena and models across spatial and temporal scales.|
|Learning Objective||4.1 The student is able to explain the connection between the sequence and the subcomponents of a biological polymer and its properties.|
The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 3.2][APLO 3.28][APLO 1.11][APLO 1.16][APLO 3.1][APLO 4.1]
Stress to students that while DNA was discovered in the 1860s, the understanding that DNA is the genetic material came only in the middle of the 20th century. Prior to that time, most scientists thought that genetic information was transmitted by proteins. There were good reasons for this belief. Scientists had observed that there were many more proteins in cells than there were DNA molecules. They also observed that the chromosomes in eukaryotic cells were packed with large protein complexes. Today scientists call those chromosomal protein complexes histones. Histones play no role in transmitting genetic information. Instead, they help package and order the chromosomes in a cell’s nucleus.
Explain the experiments demonstrating DNA’s structure and function and focus on the logic behind the findings. Ask students if they can think of alternative methods to illustrate the same findings based on what they know today.
Modern understandings of DNA have evolved from the discovery of nucleic acid to the development of the double-helix model. In the 1860s, Friedrich Miescher (Figure 14.2), a physician by profession, was the first person to isolate phosphate-rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.
To see Miescher conduct an experiment step-by-step, click through this review of how he discovered the key role of DNA and proteins in the nucleus.
- Phoebus Levene believed that the four nucleotides in DNA are not linked or repeated in the same pattern and that they are held together by phosphodiester bonds.
- He discovered that the nucleotides were held together by phosphodiester bonds, in which two phosphate groups bind two sugars together. This discovery led to our current understanding of DNA.
- He believed that proteins were less likely the vehicles for hereditary information. Later he discovered the four nucleotides in DNA which were linked together and repeated in a wide variety of different ways.
- He believed inaccurately that the four nucleotides in DNA repeated over in the same pattern. Also, he discovered that the nucleotides were held together by phosphodiester bonds in which the phosphate group binds two sugars together.
A half century later, British bacteriologist Frederick Griffith was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally,” rather than by descent. In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing morphology and physiology. He was working with Streptococcus pneumoniae, the bacterium that causes pneumonia. Griffith worked with two strains, rough (R) and smooth (S). The R strain is non-pathogenic (does not cause disease) and is called rough because its outer surface is a cell wall and lacks a capsule; as a result, the cell surface appears uneven under the microscope. The S strain is pathogenic (disease-causing) and has a capsule outside its cell wall. As a result, it has a smooth appearance under the microscope. Griffith injected the live R strain into mice and they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle (Figure 14.3). These experiments are now famously known as Griffith's transformation experiments.
Scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) were interested in exploring this transforming principle further. They isolated the S strain from the dead mice and isolated the proteins and nucleic acids, namely RNA and DNA, as these were possible candidates for the molecule of heredity. They conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.
Forensic Scientists and DNA Analysis
DNA evidence was used for the first time to solve an immigration case. The story started with a teenage boy returning to London from Ghana to be with his mother. Immigration authorities at the airport were suspicious of him, thinking that he was traveling on a forged passport. After much persuasion, he was allowed to go live with his mother, but the immigration authorities did not drop the case against him. All types of evidence, including photographs, were provided to the authorities, but deportation proceedings were started nevertheless. Around the same time, Dr. Alec Jeffreys of Leicester University in the United Kingdom had invented a technique known as DNA fingerprinting. The immigration authorities approached Dr. Jeffreys for help. He took DNA samples from the mother and three of her children, plus an unrelated mother, and compared the samples with the boy’s DNA. Because the biological father was not in the picture, DNA from the three children was compared with the boy’s DNA. He found a match in the boy’s DNA for both the mother and his three siblings. He concluded that the boy was indeed the mother’s son.
Forensic scientists analyze many items, including documents, handwriting, firearms, and biological samples. They analyze the DNA content of hair, semen, saliva, and blood, and compare it with a database of DNA profiles of known criminals. Analysis includes DNA isolation, sequencing, and sequence analysis; most forensic DNA analysis involves polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci and electrophoresis to determine the length of the PCR-amplified fragment. Also, mitochondrial DNA is sequenced for forensics. Forensic scientists are expected to appear at court hearings to present their findings. They are usually employed in crime labs of city and state government agencies. Geneticists experimenting with DNA techniques also work for scientific and research organizations, pharmaceutical industries, and college and university labs. Students wishing to pursue a career as a forensic scientist should have at least a bachelor's degree in chemistry, biology, or physics, and preferably some experience working in a laboratory.
1) Using a molecular modeling kit (or an online virtual kit such as jmol), create a model of each of the 4 nucleotides in DNA, based on structural diagrams found in this chapter or elsewhere online.
2) Identify where each nucleotide hydrogen-bonds with its complementary base. Add these bonds to secure the two pairs of nucleotides together. How does the hydrogen bonding differ between the two pairs of complementary bases?
3) Now look at a structural diagram of a complete DNA molecule. Based on the diagram, connect your two pairs of nucleotides together along your DNA’s sugar-phosphate backbone (depending on your model kit, you may have to first disconnect the hydrogen bonds between the complementary bases). Which atoms and molecules did you have to remove and add to create the sugar-phosphate backbone?
Explain why radioactive sulfur and phosphorus were used to label T2 bacteriophages in the Hershey-Chase experiments. How did the results of these experiments contribute to the identification of DNA as the genetic material?
The activity is an application of Learning Objective 4.1 and Science Practice 7.1 because students are examining the spatial relationships among the components of a DNA strand and explaining the connections between the sequence and subcomponents of the nucleotides.
An expanded lab investigation for DNA, involving restriction enzyme analysis, is available from the College Board's® AP Biology Investigative Labs: An Inquiry-Based Approach in Investigation 9.
1) Group size is dependent on the availability of molecular model kits. Please note that extra carbon and hydrogen bonds are likely needed for students to make all 4 nucleotides. Groups could also be assigned to make one of the two pairs of nucleotides, after which the groups can combine the molecules into DNA in step 3. Jmol is available for download here.
2) Note: some molecular model kits have special bonds to use for hydrogen bonds. If these bonds are not used, the students may not be able to connect the nucleotides together. Sample answer: One notable difference is that thymine and adenine bond with two hydrogen bonds while cytosine and guanine bond using three hydrogen bonds.
3) To bond two nucleotides along the sugar-phosphate backbone of DNA, we removed one H from carbon 3 of deoxyribose. We then bonded carbon 3 to one of the single-bonded O atoms on the phosphate ion. Next, we removed the OH group from the CH2 group on the deoxyribose. The carbon on this CH2 group was then bonded to the other single-bonded O on the phosphate ion.
The Think About It question is an application of Learning Objective 3.2 and Science Practice 4.1 because students are asked to justify that the Hershey-Chase experiments supported the identification of DNA as the carrier of genetic information. It is also an application of Learning Objective 3.1 and Science Practice 6.5 because students will evaluate researchers’ claims that DNA is found in cells and is the primary source of heritable information.
Experiments conducted by Martha Chase and Alfred Hershey in 1952 provided confirmatory evidence that DNA was the genetic material and not proteins. Chase and Hershey were studying a bacteriophage, which is a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA. The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages. Hershey and Chase labeled one batch of phage with radioactive sulfur, 35S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus.
Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to be detached from the host cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the tube that contained phage labeled with 35S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins (Figure 14.4).
Around this same time, Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. This is also known as Chargaff’s rules. This finding proved immensely useful when Watson and Crick were getting ready to propose their DNA double helix model.