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Chapter 16 The Molecular Basis of Inheritance DNA • Deoxyribonucleic Acid – now known to be the genetic material • function studied in microbes DNA 1928 Griffith studied Streptococcus pneumoniae -found 2 strains: smooth (encapsulated - s) & rough – r Experiment – when mice were injected with s, pathogenic - s heat killed - harmless to mice - heat killed s mixed with r - mice died - live r - harmless to mice DNA • Blood samples taken from dead mice contained live s • Somehow r cells acquired capsule ability from dead s & passed it to offspring - this phenomenon is called transformation – assimilation of external genetic material by a cell DNA 1944 Avery – determined transforming agent to be DNA 1952 Hershey & Chase – using bacteriophages (T4) which infect E. coli (to verify Avery’s work) - they knew viruses were made of DNA & protein & knew viruses cause host to make new viruses Hershey-Chase experiment • Radioactively labeled T2 with 35S mixed with bacteria, agitated in blender to separate outer phage from cells, centrifuged & measured radioactivity in supernatant Results: radioactivity in supernatant, therefore, protein did not enter the bacteria Further evidence • DNA replicates prior to cell division • Chargaff using paper chromatography analyzed various DNA samples & found DNA to be very species-specific & also very diverse DNA Structure • Polymer of nucleotides: 1) sugar (deoxyribose) backbone 2) phosphate 3) nitrogenous base: purines (adenine & guanine) pyrimidines (cytosine & thymine) Chargaff’s rules: A-T, G-C DNA Structure • 2 nucleotide strands held together by weak hydrogen bonds • X-ray crystallography showed 2 antiparallel strands of S-P wound around N-bases • Watson & Crick described structure as double helix DNA Replication • Process where DNA makes an exact copy of itself as a result of complementary base pairing: 1) molecule unwinds, then unzips (2 strands separate) due to helicase 2) new DNA nucleotides line up on both strands 3) strands recoil; continues through molecule Messelson-Stahl experiment 1) bacteria grown in 15N, then to 14N 2) DNA isolated & mixed with CsCl, centrifuged for days 3) DNA moves to areas where density equals CsCl Results: • 3 different densities indicating 3 different combinations DNA Replication • Begins at origin sites forming Y – shaped forks • helicases – unwind & single-strand binding proteins stabilize • topoisomerases – break & reseal DNA strands to allow them to unlink • primer – short segment of RNA whose base sequence is complementary to parent DNA portion needed to start new DNA segment DNA Replication • DNA polymerases – catalyze DNA synthesis 5’ 3’ • DNA ligase – links Okazaki fragments together • errors are usually removed by enzymes (natural incidence 1/10,000, completed is 1/1billion) DNA Replication • Repairs: mismatch repair – polymerase & several proteins in eukaryotes excision repair – errors cut out & removed, then replaced with correct nucleotides by DNA ligase & DNA polymerase http://www.nobel.se/medicine/educational/dna/a/replication/lagging_ani.html http://www.nobel.se/medicine/educationl/dna/a/replication/lagging ani.html :// http highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animat