Transcript Lecture 19 Spring 2011

CsCl centrifugation of DNA over time developed
by Meselson and Stahl
In class question (extra credit) for Quiz #4
Question 1: (3pts)
Why does one add EtBr to CsCl gradients for the isolation
of plasmid DNA?
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
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 2
Bacteria
Grow Faster
Why?
Why would they do 2 different growth rates?
Experiment 1
Experiment 2
N14 N15
only
N14 N15
only
Fuse Results
from
Expt 1 and 2
Cell
Divisions
N14 N15
only
Experiment 1 observations
Watson-Crick Model
N14 N15
only
Does Expt 1
prove hybrid
formation?
N15
dsDNA
N15
ssDNA
Critical
Experiment:
Hybrid Strand
Separation
And
CsCl centrifugation
Looks like
control below
What about
N14/N15 hybrid?
N14
ssDNA
N15
ssDNA
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.
Previously discussed
Frameshift Mutations
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 5Bromouracil
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 twohit kinetics.
Thymine Dimers
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.
Mutations Induced by Transposons
Wrinkled Pea
Previously discussed
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.

Mutations are induced by chemicals, ionizing irradiation,
ultraviolet light, and endogenous transposable genetic
elements.

Point mutations are of three types:
(1)
(2)
(3)
Transitions—purine for purine and pyrimidine for pyrimidine
substitutions,
Transversions—purine for pyrimidine and pyrimidine for purine
substitutions, and
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?