1、CsCl centrifugation of DNA over time developed by Meselson and Stahl,Question 1: (3pts) Why does one add EtBr to CsCl gradients for the isolation of plasmid DNA?,In class question (extra credit) for Quiz #4,Question 2: (4pts) Is an 8kb supercoiled plasmid more dense than a 3kb supercoiled plasmid. Y
2、es/No (circle one)Will an 8kb supercoiled plasmid have more EtBr bound 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 Sucrose Gradients or Agarose Gel Electrophoresis.,CsCl centrifugation of DNA over time,N15 is heavier than
3、N14-Can be resolved in CsCl,pulse-chase Experiment: Incubator with N15 containingmedium for time, then chase with N14 medium,Expt 1 grows SlowlyExpt 2 Bacteria Grow Faster Why?,Experiment 1,Experiment 2,Why would they do 2 different growth rates?,Fuse Results from Expt 1 and 2,Cell Divisions,Experim
4、ent 1 observations,Watson-Crick Model,Does Expt 1 prove hybrid formation?,Critical Experiment:Hybrid Strand Separation And CsCl centrifugation,N15 ssDNA,N14 ssDNA,N15 ssDNA,N15 dsDNA,Looks like control below,Evolution?,The Molecular Basis of Mutation-Evolution,Mutations alter the nucleotide sequence
5、s 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 Affect Base-Pairing,Mutation Caused by Tautomeric Shifts,Base Substitutions,A transition replaces a pyrimidine with anothe
6、r pyrimidine or a purine for another purine. A transversion replaces a pyrimidine with a purine or a purine with a pyrimidine.,Frameshift Mutations,Previously discussed,Factors Influencing the Rate of Spontaneous Mutations,Accuracy of the DNA replication machinery Efficiency of the mechanisms for th
7、e repair of damaged DNA Degree of exposure to mutagenic agents in the environment,Induced Mutations,Induced 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 demon
8、strated that exposing Drosophila sperm to X-rays increased the mutation frequency.,Chemical Mutagens,Types of Chemical Mutagens,Chemicals 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
9、., 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 Agents,Alkylating agents are chemicals that donate alk
10、yl 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.,Hydroxylamine,Hydroxylamine is a hydroxylating age
11、nt. Hydroxylamine hydroxylates the amino group of cytosine and leads to G:C A:T transitions.,The Electromagnetic Spectrum,X-rays induce mutations through ionization. Ultraviolet light induces mutations through excitation- Energy addition.,Irradiation Dosage and Mutation Frequency,Ionizing Radiation
12、Causes Changes in Chromosome Structure,Ionizing radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations. These types of mutations display two-hit kinetics.,Mutagenesis by Ultraviolet Irradiation,Hydrolysis of cytosine to a hydrate may cause mispairing durin
13、g replication Cross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate error-prone DNA repair mechanisms.,Thymine Dimers,Mutations Induced by Transposons,Wrinkled Pea,Expansion of Trinucleotide Repeats,Simple tandem repeats are repeated sequence of one to si
14、x nucleotide pairs. Trinucleotide repeats can increase in copy number and cause inherited diseases.Examples: Fragile X Syndrome, Huntington disease, spinocerebellar ataxiaThese diseases are characterized by anticipation, the increased severity of disease or earlier age of onset in successive generat
15、ions 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
16、, Transversionspurine for pyrimidine and pyrimidine for purine substitutions, and Frameshift 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 fluorescence to figure out-codon optimize GFP?,
