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. Yes/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

N 15 is heavier than N 14 -Can be resolved in CsCl

pulse-chase Experiment: Incubator with N 15 containing medium for time, then chase with N 14 medium Expt 1 grows Slowly Expt 2 Bacteria Grow Faster Why?

Experiment 1 Experiment 2 N 14 N 15 only Why would they do 2 different growth rates? N 14 N 15 only

Fuse Results from Expt 1 and 2 Cell Divisions N 14 N 15 only

Experiment 1 observations Watson-Crick Model Does Expt 1 prove hybrid formation? N 14 N 15 only

Critical Experiment: Hybrid Strand Separation And CsCl centrifugation N 15 ssDNA N 14 ssDNA N 15 ssDNA N 15 dsDNA Looks like control below What about N 14 /N 15 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 Affect Base-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 Transposons Wrinkled 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 fluorescence to figure out-codon optimize GFP?