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 #4
Question 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 medium
Expt 1 grows Slowly
Expt 2BacteriaGrow FasterWhy?
Experiment 1
Experiment 2
N14 N15
only
Why would they do 2 different growth rates?
N14 N15
only
Fuse ResultsfromExpt 1 and 2
Cell Divisions
N14 N15
only
Experiment 1 observations
Watson-Crick Model
Does Expt 1 prove hybrid formation?
N14 N15
only
CriticalExperiment:
Hybrid StrandSeparationAnd CsCl centrifugation N15
ssDNAN14
ssDNA
N15
ssDNA
N15
dsDNA
Looks like control below
What aboutN14/N15 hybrid?
Evolution?
The Molecular Basis of Mutation-Evolution
Mutations 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 Substitutions A 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 Mutations
Previously discussed
Factors Influencing the Rate of Spontaneous Mutations
Accuracy of the DNA replication machinery
Efficiency of the mechanisms for the 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 demonstrated 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., base analogs and acridine dyes)
A Base Analog:5-Bromouracil…it 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 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.
Hydroxylamine
Hydroxylamine is a hydroxylating agent.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 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 during 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 TransposonsWrinkled Pea
Expansion of Trinucleotide Repeats
Simple 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:(1) Transitions—purine for purine and pyrimidine for pyrimidine
substitutions,
(2) Transversions—purine for pyrimidine and pyrimidine for purine substitutions, and
(3) Frameshift mutations—additions 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 mutants…some of them
Phylogenetic relationships
How could we use GFP fluorescenceto figure out-codon optimize GFP?
