!!NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE!CLASSIC EXPERIMENTS IN MOLECULAR BIOLOGY!The Transforming Principle:!Identifying the Molecule of Inheritance!!Adapted by Howard Fried Based on an original case study by Robin Pals-Rylaarsdam, Department of Biological Science, Benedictine University, Lisle, IL ©2012 by the National Center for Case Study Teaching in Science!!!Part I – Molecule of Inheritance!!For ages it has been recognized that “traits” are passed from parents to offspring, be they humans, other animals, or plants. If there is inheritance of traits from parents to children, or from “mother cell” to “daughter cells,” then!some thing must be passed from parent to offspring. Today we know that this thing is DNA, in the form of chromosomes.!However, someone needed to figure that out!!!In the 1930s and 1940s, scientists were interested in identifying the biochemical nature of the “transforming!principle.” Fred Griffith at the British Ministry of Health observed that bacteria are capable of transferring genetic information through a process known as transformation. What did this genetic material consist of? The candidate molecules were DNA, RNA, and protein. These molecules were candidates because it was known that cell nuclei contained chromosomes that are associated with phenotypes (traits, e.g., Morgan’s fruit fly eye color!experiments wherein eye color corresponded to the X- or Y- chromosome content of the fly cells), and isolated nuclei!are composed mostly of protein, DNA, and RNA. Most scientists at the time were leaning toward protein being the!genetic material because protein is the most chemically diverse of the three types of molecules.!!Griffith was studying the bacterium Streptococcus pneumoniae, an important pathogen in the 1920s.!!Question!!1. Why would studying S. pneumoniae be an important topic in the 1920s?!!Some forms of S. pneumoniae cause disease (pneumonia),!others forms do not cause disease. When grown on a laboratory plate, one can make a good guess about the pathogenicity (disease-causing ability) of S. pneumoniae because pathogenic!strains look shiny (smooth) due to a polysaccharide!cell coat called a capsule (right side of the picture). The!capsule helps the bacteria “hide” from the immune!system long enough to cause disease. Nonpathogenic!strains lack the capsule and appear “rough” on a petri!plate (left side of the picture). Generally rough colonies!are smaller, too. Griffith used mice to “assay” the pathogenicity of the bacteria. Injecting mice with bacteria grown on petri plates either made the mice sick and killed them, or produced no disease.!!!!!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!“Classic Experiments in Molecular Biology” by Robin Pals-Rylaarsdam! Page 1!Rough!!!!!!!!!!!!!!Smooth!Image credit: Photograph by Joseph B. Haulenbeek, from Avery et al., Journal of Experimental Medicine 79 (2): 137–158. ©1944 Rockefeller University Press, used in accordance with the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license.!!!!NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE!!Question!!2. Refer to the diagram below. Fill out the table below the diagram, predicting what you think will happen as a result of each injection (mice live or mice die). !Note that “Heat-killed” indicates that the bacteria were killed at high temperature before injection into mice. When you are finished, obtain the actual results page from your TA. Compare the actual results with your answers and proceed to the questions about the results on the next page. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!“Classic Experiments in Molecular Biology” by Robin Pals-Rylaarsdam! Page 2!!!NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE!!Part II – Results!!!Questions!!1. Which of the four treatments are controls, i.e., experimental manipulations with known outcomes? Explain your answer. !!!!!!!!!!!!!!2. Which of the results is unexpected? !!!!!!3. What are two hypotheses that might explain the unexpected result? In other words, what might be going on in!the system?!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!“Classic Experiments in Molecular Biology” by Robin Pals-Rylaarsdam! Page 3!!!NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE!!Part III – Enzymes as Tools!!The properties of the rough strain are its phenotype. In the experiment you just read about, the phenotype of the living!rough strain changed from nonpathogenic to pathogenic. Scientists proposed that it changed because the genotype!changed. The next set of experiments started with the hypothesis that the genotype change was due to some extra!genetic material being added to the rough bacteria to change their phenotype to a smooth phenotype.!!The group of scientists who did the next set of experiments consisted of Oswald Avery, Colin MacLeod, and Maclyn!McCarty. Their paper was published in 1944. These scientists had three tools to use in their experiments (in addition!to the smooth and rough strains, and mice):!!1. Proteases!!!!!2. DNase!!!!!3. RNase!!!!!All three of these tools are enzymes, as you should know because their names end in “ase.”!!Questions!!1. Given the names of these three enzymes, what reaction do you expect each one to catalyze.!!!2. How could you treat dead smooth bacteria with these enzymes to determine whether DNA, RNA, or protein is!the genetic material?!!!!!!!!!!!!!!!!!!!!!!!!!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!“Classic Experiments in Molecular Biology” by Robin Pals-Rylaarsdam! Page 4!!!NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE!!Part IV – More Results!!The scientists treated heat-killed smooth bacteria with either RNase, Protease, or DNase, then combined that reaction!with living rough bacteria and injected mice with the mixture. The results are shown in the diagram.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Question!!1. What conclusion can be drawn from these data?!!!!!!!!!!!!!!!!!!!!!!!!!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !!“Classic Experiments in Molecular Biology” by Robin Pals-Rylaarsdam! Page 5!!!NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE!!What to include in your report!!1. Answers to all of the preceding questions. You may use this document to record and submit your answers. 2. Write a well-constructed essay