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Chapter 11 DNA: The Carrier of Genetic Information Experiments in DNA • ???Protein as the genetic material • 20 AA – many different combinations = unique properties • Genes control protein synthesis • DNA and RNA – only 4 nucleotides = dull Experiments in DNA • Frederick Griffith – 1928 – Bacteria – pneumococcus – 2 strains – (S) smooth strain – virulent (lethal) • Mice – pneumonia - death – (R) rough strain – avirulent • Mice survive – Heat killed (S) strain • Mice survive – Heat killed (S) + live (R) • Mice died • Found living (S) in dead mice • Griffith continued – transformation - type of permanent genetic change where the properties of 1 strain of dead cells are conferred on a different strain of living cells – “transforming principle” was transferred from dead to living cells Fig. 16-2 Mixture of heat-killed Living S cells Living R cells Heat-killed S cells and (control) (control) S cells (control) living R cells EXPERIMENT RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells • Avery, MacLeod, McCarty - 1944 – Identified Griffith’s transforming principle as DNA – Live (R) + purified DNA from (S) R cells transformed – R + (S) DNA die – R = (S) protein live – DNA responsible for transformation – Really? • Hershey and Chase – 1952 – Bacteriophages – Radioactive labels • Viral protein – sulfur • Viral DNA - phosphorus – infect bacteria, agitate in blender, centrifuge – Found • Sulfur sample – all radioactivity in supernatant (not cells) • Phosphorus sample – radioactivity in pellet (inside cells) – SO – bacteriophages inject DNA into bacteria, leaving protein on outside – DNA = hereditary material Fig. 16-3 Phage head Tail sheath Tail fiber Bacterial cell 100 nm DNA Fig. 16-4-3 EXPERIMENT Phage Empty Radioactive protein shell protein Radioactivity (phage protein) in liquid Bacterial cell Batch 1: radioactive sulfur (35S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus (32P) Centrifuge Pellet Radioactivity (phage DNA) in pellet • Rosalind Franklin (in lab of Wilkins) – X-ray diffraction on crystals of purified DNA – (X-ray crystallography) – Determine distance between atoms of molecules arranged in a regular, repeating crystalline structure • Helix structure • Nucleotide bases like rungs on ladder Fig. 16-6 (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA • James Watson and Francis Crick – 1953 – Model for DNA structure = double helix – DNA now widely accepted as genetic material – Took all available info on DNA and put together – Showed – • DNA can carry info for proteins • Serve as own template for replication Structure of DNA • Nucleotides – Deoxyribose – Phosphate – Nitrogenous base (ATCG) • Purines – adenine, guanine – 2 rings • Pyrimidines – thymine, cytosine – 1 ring – covalent bonds link = sugar-phosphate backbone • 3’ C of sugar bonded to 5’ phosphate = phophodiester linkage • 5’ end – 5’ C attached to phosphate • 3’ end – 3’ C attached to hydroxyl Chargaff - 1950 • # purines = # pyrimidines – #A = #T – #C = # G • Each cross rung of ladder – 1 purine + 1 pyrimidine Fig. 16-UN1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data • • • • Hydrogen bonding between N bases A-T = 2 H bonds G-C = 3 H bonds Complementary base pairs • # possible sequences virtually unlimited • many genes, much info Fig. 16-8 Adenine (A) Thymine (T) Guanine (G) Cytosine (C) Fig. 16-5 Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) DNA nucleotide Phosphate Sugar (deoxyribose) 3 end Guanine (G) Fig. 16-7a 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm (a) Key features of DNA structure (b) Partial chemical structure 5 end DNA Replication • Semiconservative – each strand of DNA is template to make opposite new strand • Meselson and Stahl – E. coli and isotopes of N – 15N – heavy/dense; 14N “normal” – Bacteria with 15N in DNA replicated with medium having 14N – Centrifuge – Supports semiconservative model • Explains how mutagens can be passed on Fig. 16-11a EXPERIMENT 1 Bacteria cultured in medium containing 15N 2 Bacteria transferred to medium containing 14N RESULTS 3 DNA sample centrifuged after 20 min (after first application) 4 DNA sample centrifuged after 20 min (after second replication) Less dense More dense Fig. 16-9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand Fig. 16-10 Parent cell (a) Conservative model (b) Semiconservative model (c) Dispersive model First replication Second replication Steps of DNA Replication • • • • • • 1. DNA helicase – 2. Helix-destabilizing proteins – 3. Topoisomerases – 4. RNA primer – 5. DNA polymerase – 6. Origin of replication – – Leading strand – Lagging strand • 7. DNA Ligase Leading Strand Fig. 16-12b Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0.25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes Fig. 16-14 New strand 5 end Sugar 5 end 3 end T A T C G C G G C G C T A A Base Phosphate Template strand 3 end 3 end DNA polymerase A Pyrophosphate 3 end C Nucleoside triphosphate 5 end C 5 end Fig. 16-15a Overview Origin of replication Leading strand Lagging strand Primer Leading strand Lagging strand Overall directions of replication Fig. 16-17 Overview Origin of Leading strand Lagging replication strand Singlestrand Helicase binding protein 5 3 3 Parental DNA DNA pol III PrimerPrimase 5 3 5 Leading Lagging strand Overall strand directions of Leading strand replication DNA pol III Lagging strand DNA pol I 4 35 3 2 DNA ligase1 3 5 Telomeres • Telomeres – caps end of chromosome; short non-coding sequences repeated many times • Cell can divide many times before losing crucial info • Lagging strand is discontinuous, so DNA polymerase unable to complete replication , leaving small part unreplicated small part lost with each cycle Fig. 16-19 5 Ends of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules