HC70A & SAS70A Winter 2010 Genetic Engineering in Medicine, Agriculture, and Law Professors John Harada & Bob Goldberg Lecture 2 What Are Genes & How Do They Work: Part One Course AdministratorpLast Lecture -- Age of DNA & Genetic Engineering Todayʼs Class -- What ARE Genes & How Do They Work? PART ONE Demonstration Gel Electrophoresis & Bacterial “Cloning”THEMES 1. What is the Function of a Gene? 2. What are the Properties of Genes? 3. What is the Evidence That DNA is the Genetic Material (Griffith and Avery Experiments)? 4. Is Transformation Universal? 5. What is the Structure of DNA? 6. What is the Structure of a Chromosome? 7. What is the Anatomy of a Gene? 8. What is the Colinearity Between Genes & Proteins (how does DNA→protein)? 9. How Do Switches Work to Control Gene Activity? 10. What Are the Possibilities For Manipulating Genes in the Future?Gel Electrophoresis Experiment 1. Plasmid DNA cut with restriction enzymes 2. DNA will be separated by size using gel electrophoresis 3. DNA fragments are visualized in the gel with a florescent dye.Recall: We Live in the The Age of DNA! Genetic Engineering Is Manipulating DNA! Understanding Genetic Engineering Requires a Basic Understanding of Genes And How They WorkWhat Are the Functions of a Gene?Gene Action Leads to Specific Traits – The Action of All Genes Specifies Phenotype 1. Replication 2. Gene Action 3. Cell Function (Trait) Genetic Engineering Alters Cell Function By Changing the Genotype How is this Demonstrated Experimentally? Design an Experiment! 5ʼ 3ʼ 5ʼ 3ʼ 3ʼ 5ʼ Genotype Phenotype Collection of Genes Collection of TraitsDifferent Alleles of a Gene Underlie Variations in Specific Traits Trait (e.g., Green Seeds) Alleles Mutations Lead to Different Forms of the SAME Gene (Alleles) and Generate Genetic Variability in a Population of Organisms (e.g., yellow and green peas) Genetic Engineering Can Create Infinite Amounts of Genetic Variability NOT Found in Nature (+ Rare Mutations) Mendelʼs TraitsDifferent Alleles of a Gene Can Have Different Functions Two different alleles of the Staygreen gene encode different forms of an enzyme that degrades chlorophyll Green Peas Yellow PeasBreeding Takes Advantage of Natural Genetic Variability – Gene Variability is Generated by Mutations This Genetic Variability Arose Spontaneously By RARE Mutations Gene Mutations Alter DNA Sequences, Slightly Change Gene Functions (e.g., fruit size, color), & Create Alleles -- Different Forms of the Same GeneAlleles Are Different Forms of the Same Gene That Arise By Mutation & Can be Made in a Laboratory By Modern Genetic Engineering! Alleles Alleles Reside at the Same Position on a ChromosomeHow Does the GloFish Experiment Show That Genes Direct the Production of Traits? Traits Phenotypes Different Colors!! Whatʼs Your Hypothesis?1. Replication 2. Stability (Mutations) 3. Universality a) All Cells b) All Organisms 4. Direct Cell Function/Phenotype WHAT ARE THE PROPERTIES OF A GENE? • How Can These Properties Be Tested Experimentally? • What Predictions Follow From These Properties? If DNA is the Genetic Material, THEN What……? How Show That DNA is The Genetic Material?Major Causes of Death 1920 1. Typhoid Fever 2. Malaria 3. Small Pox (virus) 4. Measles 5. Scarlet Fever 6. Whooping Cough 7. Diphtheria 8. Flu 9. Mumps 10. Cholera 2002 1. Heart Disease 2. Infectious & Parasitic Diseases 3. Cancer 4. Stroke 5. Respiratory Diseases 6. Unintended Injuries (e.g., Cars) 7. HIV/AIDS 8. Digestive Diseases 9. Diarrheal Diseases 10. Intentional Injuries (Murder, War, etc.)It is estimated that anywhere from 20 to 100 million people were killed worldwide,or the approximate equivalent of one third of the population of Europe, more than double the number killed in World War I. This extraordinary toll resulted from a high death rate of up to 50%. The Spanish Flu Pandemic - 1918 to 1920 PNAS June 6, 2000 PNAS January, 2009 By Sequencing the Virus Genome From Victims Dead For 80 Years & Synthesizing the “Original” Flu Virus By Genetic EngineeringHow Many People Have Died Worldwide During the Current H1N1 Pandemic? H1N1 Virus A. 130 B. 1,300 C. 13,000 D. 130,000 E. 1,300,000January 29, 1922 - New York City 2008 Streptococcus pneumoniae Bacterial Pneumonia Was Also a “Killer” at This Time! 1,000,000 Deaths/Year TODAY!Frederick Griffith & The Transforming Principle The First Genetic Engineering Experiment 1879-1941 Smooth Rough Streptococcus pneumoniae Invented the Word “Transformation” Not Understood For Another 50 Years Frederick GriffithA Typical Bacterial Cell Plasmids: 2,000-150,000 bp (1-100 genes) Chromosome: 500,000-5,000,000 bp (500-5,000 genes) Plasmid DNA: ~1.4 µm (10-6 m) in circumference (Genetic Engineering Vectors) Chromosome: ~ 1.4 mm (10-3 m) in circumference 2-4 µm 1-2 µm Circular Chromosome Circular Plasmid Bacterial Virus Cell Wall Cell Membrane 1 µm = 3.94 x 10-5 inchesDiversity in Bacterial Cell MorphologyTable 1-1 (part 1 of 2) Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) Bacterial Genome Projects Have Provided Remarkable Insight Into Bacterial Genomes and Cell Functions There is currently a major effort to generate The Genomic Encyclopedia of Bacteria and ArchaeaStreptococcus pneumoniae Sugar The Sugar Capsule Protects the Bacteria From Mammalian Host Antibodies Capsule CapsuleStreptococcus pneumoniae Genome Has Been Sequenced! 2,046,115 bp and 1,987 Genes J. Bacteriology 2001 Note Genes on BOTH DNA Strands! Virulence CapsuleThe Griffith Experiment With Smooth and Rough Pneumonia Bacteria Smooth Virulent Form Rough Avirulent Form Smooth Rough Spontaneous Mutations J. Hygiene, 1928 virulent avirulentThe Griffiths Experiment LIVE Rough Cells TRANSFORMED by DEAD Smooth Cells!!! HOW? What Was the Transforming Principle? Hypothesis? 1 2 3 4What is the Transforming Principle? Experiments of Avery, McCleod, & McCarty Fast Forward to the 1940s! Oswald Avery 1877-1955 Colin McCleod 1909-1972 Macyln McCarty 1911-2005 J. Exp. Med.,1944 DNA is the Genetic Material! One of the Major Reasons Watson and Crick Considered DNA As the Genetic Material In Order to Solve DNA Structure1. Does the Transforming Principle Come From the Mouse or Bacteria? 2. If From the Bacteria -- What Component? 3. How