cscl centrifugation of dna over time developed by meselson and stahl

Click here to load reader

Post on 03-Jan-2016

220 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • CsCl centrifugation of DNA over time developedby Meselson and Stahl

  • Question 1: (3pts)Why does one add EtBr to CsCl gradients for the isolationof plasmid DNA? In class question (extra credit) for Quiz #4Question 2: (4pts)Is an 8kb supercoiled plasmid more dense than a3kb supercoiled plasmid. Yes/No (circle one)

    Will an 8kb supercoiled plasmid have more EtBrbound to it? Yes/No (circle one)

  • We will talk about this again in a later lecture:But CsCl gradients are not the same thing as SucroseGradients or Agarose Gel Electrophoresis.

  • CsCl centrifugation of DNA over time

  • N15 is heavier than N14-Can be resolved in CsCl

  • pulse-chase Experiment: Incubator with N15 containing medium for time, then chase with N14 mediumExpt 1 grows Slowly

    Expt 2BacteriaGrow FasterWhy?

  • Experiment 1Experiment 2Why would they do 2 different growth rates?

  • Fuse ResultsfromExpt 1 and 2

    Cell Divisions

  • Experiment 1 observationsWatson-Crick ModelDoes Expt 1 prove hybrid formation?

  • CriticalExperiment:

    Hybrid StrandSeparationAnd CsCl centrifugationN15ssDNAN14ssDNAN15ssDNAN15dsDNALooks like control below

  • Evolution?

  • The Molecular Basis of Mutation-EvolutionMutations alter the nucleotide sequences of genes in several ways, for example the substitution of one base pair for another or the deletion or addition or one or a few base pairs.

  • Tautomeric Shifts

  • Tautomeric Shifts AffectBase-Pairing

  • Mutation Caused by Tautomeric Shifts

  • Base SubstitutionsA transition replaces a pyrimidine with another pyrimidine or a purine for another purine.A transversion replaces a pyrimidine with a purine or a purine with a pyrimidine.

  • Frameshift MutationsPreviously discussed

  • Factors Influencing the Rate of Spontaneous MutationsAccuracy of the DNA replication machineryEfficiency of the mechanisms for the repair of damaged DNADegree of exposure to mutagenic agents in the environment

  • Induced MutationsInduced mutations occur upon exposure to physical or chemical mutagens.Hermann J. Muller and Edgar Alternburg measured the frequency of X-linked recessive lethal mutations in Drosophila.Muller demonstrated that exposing Drosophila sperm to X-rays increased the mutation frequency.

  • Chemical Mutagens

  • Types of Chemical MutagensChemicals that are mutagenic to both replicating and nonreplicating DNA (e.g., alkylating agents and nitrous acid)Chemicals that are mutagenic only to replicating DNA (e.g., base analogs and acridine dyes)

  • A Base Analog:5-Bromouracilit is more like Thymine!

  • Mutagenic Effects of 5-Bromouracil

  • Nitrous Acid Causes Oxidative Deamination of Bases

  • Intercalation of an Acridine Dye Causes Frameshift Mutations

  • Alkylating AgentsAlkylating agents are chemicals that donate alkyl groups to other molecules.Alkylating agents induce transitions, transversions, frameshifts, and chromosome aberrations.Alkylating of bases can change base-pairing properties.Alkylating agents can also activate error-prone DNA repair processes.

  • HydroxylamineHydroxylamine is a hydroxylating agent.Hydroxylamine hydroxylates the amino group of cytosine and leads to G:C A:T transitions.

  • The Electromagnetic SpectrumX-rays induce mutations through ionization.Ultraviolet light induces mutations through excitation- Energy addition.

  • Irradiation Dosage and Mutation Frequency

  • Ionizing Radiation Causes Changes in Chromosome StructureIonizing radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations.These types of mutations display two-hit kinetics.

  • Mutagenesis by Ultraviolet IrradiationHydrolysis of cytosine to a hydrate may cause mispairing during replicationCross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate error-prone DNA repair mechanisms.Thymine Dimers

  • Mutations Induced by TransposonsWrinkled Pea

  • Expansion of Trinucleotide RepeatsSimple tandem repeats are repeated sequence of one to six nucleotide pairs.Trinucleotide repeats can increase in copy number and cause inherited diseases.

    Examples: Fragile X Syndrome, Huntington disease, spinocerebellar ataxia

    These diseases are characterized by anticipation, the increased severity of disease or earlier age of onset in successive generations as the trinucleotide copy number increases.Previously discussed

  • Mutations are induced by chemicals, ionizing irradiation, ultraviolet light, and endogenous transposable genetic elements.

    Point mutations are of three types:Transitionspurine for purine and pyrimidine for pyrimidine substitutions,Transversionspurine for pyrimidine and pyrimidine for purine substitutions, andFrameshift mutationsadditions or deletions of one or two nucleotide pairs, which alter the reading frame of the gene distal to the site of the mutation.You must know for your future

  • Evolution?

  • Alpha and Beta chain mutantssome of them

  • Phylogenetic relationships

  • How could we use GFP fluorescenceto figure out-codon optimize GFP?

    ***********************************************