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History Fred Griffith – 1928 Streptococcus pneumonia – bacterial pneumonia Two strains Smooth (S) – capsule: virulent pathogen Rough (R) – non-virulent S mouse died R mouse lived Heat-killed S + R mouse died blood (smooth live bacteria) Transforming principle Griffith’s Transformation Experiment History Friedrich Miescher – 1869 Isolated DNA T.H. Morgan Genes located on chromosomes DNA and proteins became candidates for genetic material Proteins took the lead: heterogeneity and specific function History Max Delbrück and Salavador Luria – 1940 Bacteriophage (bacteria eaters) E. coli phages T1-T7 Easy to work with, reproduce every 25 minute Oswald Avery 1944 Purified various chemicals from pathogenic bacteria; tried to transform live non-virulent bacteria DNA is the transforming principle Greeted with skepticism History Erwin Chargaff – 1950 DNA composition varies from one species to another % ratio of nucleotides (A=T and G = C) Hershey and Chase – 1952 Blender experiment S-32 - proteins P-35 - DNA Blender Experiment Blender Experiment Blender Experiment Watson and Crick Nature: 1953 X-ray crystallography - Franklin/Wilkens Helical shape, width of molecule, composed of two strands (double helix) Chargaff Base Pairing Rule DNA Structure Nucleotide: building block (Monomer) 5-carbon sugar: deoxyribose Phosphate group Nitrogenous base Adenine, Guanine, Thymine, Cytosine Antiparallel chains run in opposite directions DNA Molecule Double Helix Antiparallel chains run in opposite directions 5′ end phosphate attached to 5′ deoxyribose carbon 3′ end hydroxyl attached to 3′ deoxyribose carbon Base Pairing Hydrogen bonding • Occurs between base pairs; binds the two DNA strands • Adenine (A) with thymine (T) form two hydrogen bonds • Guanine (G) with cytosine (C) form three hydrogen bonds DNA Replication Meselson-Stahl Experiment Semi-conservative replication • E. coli grown in medium containing heavy nitrogen (15N): incorporated 15N into DNA Transferred from 15N to 14N medium after two generations, DNA density supported semiconservative replication Semiconservative replication Parental DNA First generation Second generation Fig. 12-7a, p. 268 Semi-conservative Replication Each daughter double helix consists of 1 original strand from parent molecule 1 new complementary strand Adenine Deoxyribose Guanine Deoxyribose Thymine Deoxyribose Cytosine Deoxyribose Fig. 12-6b, p. 267 DNA Replication Origins of Replication - - specific nucleotide sequence where replication begins Attachment site for proteins that initiate replication – forms a replication bubble Replication is bi-directional Eukaryotic cells have multiple origins of replication -Strands separate - Helicases: open the double helix - Single-strand binding proteins (SSB): keep strands separated and stabilize DNA Bi-directional Replication DNA Replication Synthesis of new strand: - DNA polymerase: catalyzes elongation of DNA strand - adds nucleotides to the free 3/ end; DNA strand grows in a 5/ to 3/ direction - 53 Rule - energy provided by nucleoside triphosphate DNA Replication Always proceeds in 5′ → 3′ direction Leading strand synthesized continuously Lagging strand synthesized discontinuously forms short Okazaki fragments (100-200 nucleotides) DNA primase synthesizes RNA primers DNA ligase links Okazaki fragments DNA Replication Priming DNA polymerase can only add to a pre-existing chain – which is the RNA primer Short RNA strand (5-14 nucleotides) Removed later