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?