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CH_6_student_outline - 0 DNA How the molecule of heredity...

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Unformatted text preview: 0 DNA How the molecule of heredity carries, replicates, and recombines information Chapter 6 0 I. Experiments designate DNA as the genetic material A. Chemical characterization localizes DNA in the chromosomes 0 1869, Miescher investigated the chemical composition of the nucleus Extracted a weakly acidic, phosphorous rich material from nuclei of human white blood cells He called it "nuclein" We call it DNA (deoxyribonucleic acid) today Deoxyribose a sugar; acidic Four subunits belonging to class of compounds called nucleotides linked together by phosphodiester bonds Chromosomes are composed of DNA The chemical composition of DNA 0 sugar-phosphate "backbone" sugar is a 5 carbondeoxyribose nucleotides or bases face inwards Are genes composed of DNA or protein? 0 DNA Only four different subunits make up DNA. Chromosomes contain less DNA than protein by weight. Protein 20 different subunits greater potential variety of combinations Chromosomes contain more protein than DNA by weight. B. Bacterial transformation implicates DNA as the substance of genes 0 1928 Griffith Attempting to develop a vaccine Isolated two strains of Streptococcus pneumoniae Rough strain (R) was harmless Smooth strain (S) was pathogenic 0 Griffith experiment 0 Griffith experiment Griffith experiment (better summary) 1. Mice injected with live cells of harmless strain R. 2. Mice injected with live cells of killer strain S. 3. Mice injected with heat-killed S cells. 4. Mice injected with live R cells plus heat-killed S cells. 0 R S dead S R+ dead S X X 0 1. Transformation What happened in the fourth experiment? The harmless R cells had been transformed by material from the dead S cells Descendents of the transformed cells were also pathogenic 0 Avery, MacLeod, McCarty experiment 0 1944 Avery, MacLeod, and McCarty determined that DNA is the transformation material So, what exactly was the transforming material? Cell extracts treated with protein-digesting enzymes could still transform bacteria Cell extracts treated with DNA-digesting enzymes lost their transforming ability Concluded that DNA, not protein, transforms bacteria Avery, MacLeod, McCarty experiment 2. Bacterial transformation is caused by DNA 0 Avery, MacLeod, McCarty experiment 2. Bacterial transformation is caused by DNA 0 0 C. Hershey and Chase experiments 1952 Hershey & Chase provide convincing evidence that DNA is genetic material Waring blender experiment using T2 bacteriophage and bacteria Created labeled bacteriophages Radioactive sulfur S35 labeled proteins Radioactive phosphorus P32 labeled DNA Allowed labeled viruses to infect bacteria Asked: Where are the radioactive labels after infection? Inside or outside cell? 0 Bacteriophages Viruses that infect bacteria Consist of protein coat and DNA core Inject their hereditary material into bacteria Experiment examined what was transforming part, protein or DNA? bacterial cell wall plasma membrane cytoplasm Hershey and Chase Waring blender experiment 0 Hershey and Chase Waring blender experiment 0 virus particle labeled with 35S virus particle 0 labeled with 32P Hershey & Chase Results S35 labeled proteins P32 labeled DNA core bacterial cell (cutaway view) label outside cell label inside cell II. The Watson-Crick Model: DNA is a double helix 0 1951 Watson learns about x-ray diffraction pattern projected by DNA Knowledge of the chemical structure of nucleotides (deoxyribose sugar, phosphate, and nitrogenous base) Chargaff's experiments demonstrate that ratio of A and T are 1:1, and G and C are 1:1 1953 Watson & Crick propose their double helix model of DNA structure X-ray diffraction patterns produced by DNA0 fibers Rosalind Franklin and Maurice Wilkins A. DNA's chemical constituents 1. Deoxyribose sugar. 3. Four nitrogenous bases Purines 0 A. DNA's chemical constituents Assembly into a nucleotide 0 0 DNA's chemical constituents 0 Chargaff's ratios Complementary base pairing by formation of hydrogen bonds explain Chargaff's ratios 0 GC pair : 3 H bonds AT pair : 2 H bonds B. DNA structure 0 DNA is double helix Strands are anti-parallel (5' 3' & 3' 5') with a sugar-phosphate backbone on outside and pairs of bases in the middle Two strands wrap around each other every 30 Angstroms, once every 10 base pairs Two chains are held together by hydrogen bonds between A-T and G-C base pairs Structurally, purines (A and G) pair best with pyrimadines (T and C) Thus, A pairs with T and G pairs with C, also explaining Chargaff's ratios 0 C. Double helix may assume alternative forms 0 0 Some DNA molecules are circular instead of linear Prokaryotes Mitochondria Chloroplasts Viruses Some viruses carry single-stranded DNA bacteriophages AIDS Some viruses carry RNA D. Four requirements for DNA to be genetic material 0 Must carry information Cracking the genetic code DNA replication Mutation Gene function Must replicate Must allow for information to change Must govern the expression of the phenotype III. DNA stores information in the sequence of0its bases A. Much of DNA's sequence-specific information is accessible only when the double helix is unwound - Proteins read the DNA sequence of nucleotides as the DNA helix unwinds. Proteins can either bind to a DNA sequence, or initiate the copying of it. - Human genome is believed to be 250 million nucleotides long. Four possible nucleotides. Thus 4250,000,000 possible sequences in the human genome. - An average single coding gene sequence might be about 10,000 bases long. Thus, 410,000 possibilities for an average gene. B. Some genetic information is accessible even in intact, double-stranded DNA molecules - Some proteins recognize the base sequence of DNA without unwinding it - One example is a restriction enzyme. C. Some viruses use RNA as the repository of genetic information 0 IV. DNA replication: Copying genetic information for transmission to the next generation 0 Complementary base pairing produces semiconservative replication. Double helix unwinds Each strand acts as template Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C. Two daughter helices produced after replication 0 Experimental proof of semiconservative replication three possible models 0 Semiconservative replication Watson and Crick model Conservative replication: The parental double helix remains intact; both strands of the daughter double helix are newly synthesized. Dispersive replication: At completion, both strands of both double helices contain both original and newly synthesized material. 0 0 CONSERVATIVE Would somehow produce an entirely new DNA strand during replication and 2 parental strands reassociate together again 0 DISPERSIVE Reassembly of molecules that were a mix of old and new fragment sections, but not complete strands 0 SEMI-CONSERVATIVE New DNA would consist of one new and one old strand of DNA, without disrupting strand integrity Meselson-Stahl experiments confirm semi-conservative replication 0 Fig. 6.16 0 The mechanism of DNA replication Arthur Kornbuerg, a nobel prize winner and other biochemists deduced steps of replication. Initiation Proteins bind to DNA and open up double helix. Prepare DNA for complementary base pairing Elongation Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA. 0 0 0 Enzymes involved in replication Pol III produces new stands of complementary DNA Pol I fills in gaps between newly synthesized Okazaki segments DNA helicase unwinds double helix Single-stranded binding proteins keep helix open Primase creates RNA primers to initiate synthesis Ligase welds together Okazaki fragments Pol II proofreading of the newly synthesized DNA 0 Replication is bidirectional Replication forks move in opposite directions. In linear chromosomes, telomeres ensure the maintenance and accurate replication of chromosome ends In circular chromosomes, such as E. coli, there is only one origin of replication. In circular chromosomes, unwinding and replication causes supercoiling, which may impede replication. Topoisomerase enzyme that relaxes supercoils by nicking strands The bidirectional replication of a circular chromosome 0 0 Cells must ensure accuracy of genetic information 0 Redunancy Basis for repair of errors that occur during replication or during storage Enzymes repair chemical damage to DNA. Errors during replication are rare. V. Recombination reshuffles the information content of DNA 0 During recombination, DNA molecules break and rejoin. Meselson and Weigle - Experimental evidence from viral DNA and radioactive isotopes Co-infected E. coli with light and heavy strains of virus after allowing time for recombination Separated on a CsCl density gradient Meselson and Weigle demonstrate recombination occurs by breakage and rejoining of DNA 0 Heteroduplexes mark the spot of recombination 0 Products of recombination are always in exact register; not a single base pair is lost or gained. Two strands do not break and rejoin at the same location; often they are hundreds of base pairs apart. Region between break points is called heteroduplex. 0 Heteroduplex region Double stranded break model of meiotic recombination 0 Homologs physically break, exchange parts, and rejoin Breakage and repair create reciprocal products of recombination Recombination events can occur anywhere along the DNA molecule Precision in the exchange prevents mutations from occurring during the process Double stranded break formation 0 spoI protein breaks one chromatid on both strands Resection 0 5' end on each side of break are degraded to produce two 3' single stranded tails First strand invasion 0 RecA binds 3' tail and double helix allowing invasion and migration Formation of Holliday junctions 0 New DNA synthesis forms two X structures called Holliday junctions Branch migration Both invading strands zip up and migrate while newly created heteroduplex molecules rewind behind. 0 The Holliday intermediate 0 Interlocked non-sister chromatids disengage. Two resolutions are possible Alternative resolutions Endonuclease cuts Holliday intermediate 0 ...
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