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The Molecular Basis of Inheritance Figuring out the genetic material of organisms is made of DNA, not protein… Frederick Griffith (1928)Experiment:-living S cells (control) injected into mouse – dies-living R cells (control) injected into mouse – lives-heat-killed S cells (control) injected into mouse – lives-mixture of heat-killed S and living R cells – dies – has living S cells inside-conclusion: living R bacteria transformed into deadly S bacteria by unknown, heritable substance. Oswald Avery, et al. (1944)-discovered that the transforming agent was DNA Hershey and Chase (1952)-bacteriophages: virus that infects bacteria, composed of DNA and protein-protein radiolabel S, DNA radiolabel P-conclusion: radiolabeled DNA entered infected bacteria à DNA must be the genetic material Edwin Chargaff (1947) – Chargaff’s Rules:-DNA composition varies between species-ratios: %A = %T and %G = %C Rosalind Franklin (1950s)-worked with Maurice Wilkins-X-ray crystallography = images of DNA-provided measurements on chemistry of DNA James Watson and Francis Crick (1953)-discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s rules Structure of DNA-DNA = double helix-backbone = sugar + phosphate-rungs = nitrogenous bases-nitrogenous bases: purines = Adenine (A), Guanine (G) pyrimidines = Thymine (T), Cytosine (C)-pairing: purine + pyrimidine (A double Hbonds with T, G triple Hbonds with C)-hydrogen bonds between base pairs of the 2 strands hold the molecule together like a zipper-DNA is antiparallel – one strand runs 5’à3’, other strand runs opposite 3’à5’ (upside down)-from small to large: DNA double helix, wrap around histone proteins to create nucleosomes (beads on a string), which coil into the 30nm fiber, which loops on scaffolding into a 300nm fiber which winds into a chromatid, half of a replicated chromosome. DNA Comparison (prokaryotic vs. eukaryotic)Prokaryotic – double-stranded, circular, one chromosome, in cytoplasm, no histones, supercoiled DNAEukaryotic – double-stranded, linear, usually 1+ chromosomes, in nucleus, DNA wrapped around histone proteins, forms chromatin How DNA ReplicatesReplication – making DNA from existing DNA-Meselson and Stahl experiment: bacteria cultured in medium with 15N (heavy isotope) à bacteria transferred to medium with 14N (lighter isotope) à DNA sample centrifuged after 1st replication à DNA sample centrifuged after 2nd replication-conclusion: 1st replication all the same, 2nd replication some DNA less dense and some half way dense (not totally heavy) à thus replication is semiconservative-semiconservative: parent molecule separates à separation of strands à daughter DNA molecules each consisting of one parental strand and one new strand Major Steps of DNA Replication:1. Helicase: unwinds DNA at origins of replicationa. Single-strand binding protein (SSBp) – binds to and stabilizes single-stranded DNA until it can be used as a templateb. Topoisomerase – relieves “overwinding” strain ahead of replication forks by breaking, swiveling, and rejoining DNA strands.2. Initiation proteins separate 2 strands à forms replication bubble/replication forks3. Primase: puts down RNA primer to start replicationa. Makes the RNA primer at 5’ end of leading strand and each of the Okazaki fragments of lagging strand4. DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’à3’). DNApolIII only works 5’à3’a. Uses parental DNA as template, makes new DNA strand by adding nucleotides to 3’ end of a preexisting DNA strand or primer5. Lagging strand grows in 3’à5’ direction by the addition of Okazaki fragments6. DNA polymerase I: replaces RNA primers with DNA7. DNA ligase: seals fragments togethera. Joins 3’ end of DNA that replaces primer to rest of leading strand and joins Okazaki fragments of lagging strand Leading strand vs. lagging strandLeading – continuousLagging – in pieces since DNApolII only works in the one direction.-forms Okazaki fragmants – short segments of DNA that grow 5’à3’ that are added onto the lagging strand -DNA ligase seals them together Proofreading and Repair-DNA polymerases proofread as bases are added-mismatch repair: special enzymes fix incorrect pairings-nucleotide excision repair: nucleases cut damaged DNA, then DNApol and ligase fill in the gaps-errors: pairing errors – 1 in 100,000 nucleotides. Complete DNA – 1 in 10 billion nucleotides Problem at the 5’ end-DNA polymerase only adds nucleotides to the 3’ end-no way to complete 5’ ends of daughter strands-over many replications, DNA strands will grow shorter and shorter Telomeres = repeated units of short nucleotide sequences (TTAGGG) at the ends of DNA-telomeres cap the ends of DNA to postpone erosion of genes at ends (TTAGGG)-telomerase – enzyme that adds to telomeres – eukaryotic germ cells, cancer cells -telomerase extends the 3’ end of a DNA strand -the other strand is extended in the usual way by promise, DNApol, and ligase -the result is a longer telomere with a 3’end overhang
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