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The Hereditary Material Advanced Douglas Wilkin, Ph.D. Say Thanks to the Authors Click http://www.ck12.org/saythanks (No sign in required) To access a customizable version of this book, as well as other interactive content, visit www.ck12.org CK-12 Foundation is a non-profit organization with a mission to reduce the cost of textbook materials for the K-12 market both in the U.S. and worldwide. Using an open-source, collaborative, and web-based compilation model, CK-12 pioneers and promotes the creation and distribution of high-quality, adaptive online textbooks that can be mixed, modified and printed (i.e., the FlexBook® textbooks). Copyright © 2016 CK-12 Foundation, www.ck12.org The names “CK-12” and “CK12” and associated logos and the terms “FlexBook®” and “FlexBook Platform®” (collectively “CK-12 Marks”) are trademarks and service marks of CK-12 Foundation and are protected by federal, state, and international laws. Any form of reproduction of this book in any format or medium, in whole or in sections must include the referral attribution link http://www.ck12.org/saythanks (placed in a visible location) in addition to the following terms. Except as otherwise noted, all CK-12 Content (including CK-12 Curriculum Material) is made available to Users in accordance with the Creative Commons Attribution-Non-Commercial 3.0 Unported (CC BY-NC 3.0) License (http://creativecommons.org/ licenses/by-nc/3.0/), as amended and updated by Creative Commons from time to time (the “CC License”), which is incorporated herein by this reference. Complete terms can be found at http://www.ck12.org/about/ terms-of-use. Printed: November 9, 2016 AUTHOR Douglas Wilkin, Ph.D. www.ck12.org C HAPTER Chapter 1. The Hereditary Material - Advanced 1 The Hereditary Material Advanced • Discuss how the work of Griffith, Avery, Hershey, and Chase demonstrated that DNA is the genetic material. • Define transformation and explain that transformation is the change in genotype and phenotype due to the assimilation of the external DNA by a cell. DNA or Protein? Which is the hereditary material? 100 years ago, it was not known. Some thought it was protein, others thought it was DNA. This image actually represents both DNA and protein, in the form of the enzyme DNA polymerase I, an enzyme involved in DNA replication. Here it is shown interacting with DNA. The Hereditary Material For almost 100 years, scientists have known plenty about proteins. They have known that proteins of all different shapes, sizes, and functions exist. For this reason, many scientists believed that proteins were the heredity material. It wasn’t until 1928, when Frederick Griffith identified the process of transformation, that individuals started to question this concept. Griffith demonstrated that transformation occurs, but what was the material that caused the transforming process? Frederick Griffith Griffith was studying Streptococcus pneumoniae, a bacterium that infects mammals. He used two strains of this bacteria, a virulent S (smooth) strain and a harmless R (rough) strain to demonstrate the transfer of genetic material. 1 www.ck12.org The S strain is surrounded by a polysaccharide capsule, which protects it from the host’s immune system, resulting in the death of the host, while the R strain, which does not have the protective capsule, is defeated by the host’s immune system. Hence, when mammalian cells are infected with the R strain bacteria, the host does not die (Figure 1.1). Griffith infected mice with heat-killed S strain bacteria. As expected, the heat-killed bacteria, as they were dead, had no effect on the mice. But then he tried a novel experiment. He mixed the remains of heat-killed S strain bacteria with live R strain bacteria and injected the mixture into mice. Remember, separately both of these bacteria are harmless to the mice. And yet the mice died (Figure 1.1). Why? These mice had both live R and live S strain bacteria in their blood. How? Griffith concluded that the R strain had changed, or transformed, into the lethal S strain. Something, such as the "instructions” from the remains of the S strain, had to move into the R strain in order to turn the harmless R strain into the lethal S strain. This material that was transferred between strains had to be the heredity material. But the transforming material had yet to be identified. Transformation is now known as the change in genotype and phenotype due to the assimilation of external DNA (heredity material) by a cell. FIGURE 1.1 Griffith’s Experimental Results. Griffith showed that a substance could be transferred to harmless bacteria and make them deadly. The rough (R) strain has no effect on the mouse, whereas the smooth (S) strain is harmful to the mouse. Heatkilled S strain also has no effect on the mouse, but the mixture of heat-killed S strain and the R strain is harmful to the mouse. Oswald Avery and Colleagues Over the next decade, scientists, led by Oswald Avery, tried to identify the material involved in transformation. Avery, together with his colleagues Maclyn McCarty and Colin MacLeod, removed various organic compounds from S strain bacteria and tested the remaining compounds for the ability to cause transformation. If the remaining material did not cause transformation, than that material could not be the heredity material. Avery and his colleagues treated the S strain bacteria with the protease enzymes trypsin and chymotrypsin, or ribonuclease or deoxyribonuclease, mixed the remaining extract with R strain bacteria, and asked if transformation of the R strain bacteria still occurred. They found that the proteases, which remove proteins from cells, and ribonuclease, which break apart RNA, did not affect transformation, but an enzyme preparation of deoxyribonuclease, which breaks down DNA, destroyed the transforming power. Together, these results showed that neither proteins nor RNA carried the "instructions" (or the genes) which allowed transformation. These results also indicated that DNA was the heredity material. The year was 1944. However, this finding was not widely accepted, partly because so little was known about DNA. It was still thought that proteins were better candidates to be the heredity material. The structure of DNA was still unknown, and many scientists were not convinced that genes from bacteria and more complex organisms could be similar. See A gene is made of DNA at http://www.dnaftb.org/17/animation.html to see Oswald Avery describe his Griffith’s and his work. 2 www.ck12.org Chapter 1. The Hereditary Material - Advanced TABLE 1.1: Avery-MacLeod-McCarty Experiment Treatment proteases ribonuclease deoxyribonuclease Destroys protein RNA DNA Transformation yes yes no Alfred Hershey and Martha Chase In 1952, Alfred Hershey and Martha Chase put this skepticism to rest. They conclusively demonstrated that DNA is the genetic material. Hershey and Chase used the T2 bacteriophage, a virus that infects bacteria, to prove this point. A virus is essentially DNA (or RNA) surrounded by a protein coat (Figure 1.2). To reproduce, a virus must infect a cell and use that host cell’s machinery to make more viruses, a replicative process known as the lytic cycle. The T2 bacteriophage can quickly turn an Escherichia coli (E. coli) bacteria into a T2 producing system. But to do that, the genetic material from T2, which could only be either protein or DNA, must be transferred to the bacteria. Which one was it? FIGURE 1.2 Structural overview of T2 phage. A 2- dimensional representation is on the left, and a 3-dimensional representation is on the right. The phage is essentially nucleic acid surrounded by a protein coat. Hershey and Chase performed a series of classic experiments, taking advantage of the fact that T2 is essentially just DNA and protein. In the experiments, T2 phages with either radioactive 32 P-labeled DNA or radioactive 35 Slabeled protein were used to infect bacteria. Either the radioactive proteins or radioactive DNA would be transferred to the bacteria, depending on which one was the genetic material. Thus, identifying which one was transferred would identify the genetic material. In both experiments, bacteria were separated from the phage coats by blending, followed by centrifugation. Hershey and Chase then asked which phase following centrifugation was radioactive: the supernatant or the cell pellet. They also asked what was the radioactivity in the bacterial pellet: 32 P or 35 S. Only the radioactively labeled DNA was found inside the bacteria, whereas the radioactive proteins stayed in the solution (Figure 1.3). These experiments demonstrated that DNA is the genetic material and that protein does not transmit genetic information. Summary • The work of several researchers led to the discovery that DNA is the genetic material. 3 www.ck12.org FIGURE 1.3 The Hershey and Chase experiment. T2 virus with either radioactive protein (upper section) or radioactive DNA (lower section) were used to infect bacteria. A blender was used to remove the phage from the bacteria followed by centrifugation. The radioactive DNA was found inside the bacteria (lower section), demonstrating that DNA is the genetic material. • Along the way, Griffith discovered the process of transformation. Avery discovered that DNA was the hereditary material. Hershey and Chase confirmed that DNA is the genetic material. Review 1. Outline research that determined that DNA is the genetic material. 2. What is transformation? References 1. Mariana Ruiz Villarreal (LadyofHats) for CK-12 Foundation. CK-12 Foundation . CC BY-NC 3.0 2. Laura Guerin. CK-12 Foundation . CC BY-NC 3.0 3. Laura Guerin. CK-12 Foundation . CC BY-NC 3.0 4