Transcript Chromosome structure

Announcements
1. http://cwx.prenhall.com/bookbind/pubbooks/klug3/
Self-grading problems = good practice for exam
Review of Last Lecture
1. Post-transcriptional gene regulation: Alternative splicing
2. Classification of mutations
3. Detection of mutations in humans
4. Different forms of mutations
Outline of Lecture 29
I. Origins of mutation
II. Mechanisms of DNA repair
III. Transposable elements
I. Different origins of mutation
1. Tautomeric Shifts: spontaneous
2. Base Analogues: chemical
3. Alkylating Agents: chemical
4. Intercalating Dyes: chemical
environmental
5. Deamination: chemical
6. UV Radiation and Thymine Dimers
7. High-Energy Radiation (X rays, gamma rays, cosmic
rays)
Mispairing Due to Tautomeric Shifts
Formation of a TA to CG Transition
During DNA Replication
Transition is a purine replaced by different purine or pyrimidine
replaced by different pyrimidine.
2. Base Analogues: DNA can
Incorporate 5-BU in place of Thymine
common
rare
Changes T-A pair > C-G pair. T > C, and A > G are both Transitions
3. Alkylating Agents: Ethylmethane
Sulfonate (EMS) Alkylates Guanine
Note: changes a G-C pair into an A-T pair
(G > A is a transition, C > T is a transition)
Another example: mustard gases first used in WWII.
4. Intercalating Dyes Cause Frameshifts
Intercalate themselves into the DNA double helix, distorting it,
and causing insertion or deletion during DNA replication or
recombination. Other examples: Ethidium Bromide, DAPI.
5. Deamination is Caused by Nitrous Acid
(a) Causes: C -> U/T transition (and G -> A transition)
(b) Causes: A -> G transition (and T -> C transition).
Deamination can be spontaneous as well.
6. Ultraviolet Radiation Cause
Thymine Dimers
260 nanometer
wavelength
Disrupts synthesis;
good for sterilization
of bacteria, bad for
skin cancer.
7. High-Energy (Ionizing) Radiation
Effects of Ionizing Radiation
• Causes either point mutations or breaks in
phosphodiester bonds of DNA backbone.
• If both strands broken, there can sometimes be repair
in mammals through the double-strand break repair
(DSB) system.
• Dividing cells are more susceptible to therapeutic Xrays than non-dividing cells (radiation therapy for
cancer).
Example of ionizing radiation
1986 - nuclear reactor in Chernobyl, Soviet Union overheated,
exploded, and ejected radioactive material into the
environment - largest radiation accident in world.
Gamma rays emitted from radioactive elements are a source
of ionizing radiation: 31 killed, 200 + acute radiation sickness,
longterm effects ???
No increase so far in # of leukemias, but significant
increase in # of childhood thyroid cancers
normal - 0.5-3 cases / million children
now- 100 cases / million children
Ionizing radiation transforms stable atoms into reactive free
radicals and ions, which cause mutations in DNA
Radiation Doses in Perspective
Category
Lethal full-body dose
Detectable increase in cancer
Chernobyl cleanup worker
Chernobyl nearby resident
Germany resident
Average yearly medical
Yearly background
Yearly smoking
Dose (mSv, millisievert)
3000
> 200
250
50
0.4-0.9
0.39
2-3
2.8
Irradiation of Food (and Mail?)
• Food/mail is exposed to X rays or electrons; doesn’t
contact radioactive material or become radioactive itself.
• Pro:
– Prevents spoilage of food.
– Reduces bacterial food-borne diseases.
– Reduces reliance on chemical preservatives.
• Con:
– Produces chemical changes in food (so do other
preservation methods); safe?
– Selects for radiation-resistant bacteria?
II. Mechanisms of DNA Repair
• Prokaryotic:
– Photoactivation Repair
– Base Excision Repair
– Post-replication Repair and SOS Repair
• Eukaryotic:
– Nucleotide Excision Repair
– Proofreading and Mismatch Repair
– Double-Strand Break Repair
Photoactivation Repair in Prokaryotes
UV
Photoreactivation
Enzyme Cleaves
bond between T’s
Visible light
Base Excision Repair in Prokaryotes
DNA glycosylase
Apyrimidinic endonuclease
DNA Pol I, DNA ligase
Nucleotide Excision Repair:
Prokaryotes and Eukaryotes
uvr nuclease
DNA Pol I
DNA ligase
Model for Eukaryotic Nucleotide Excision
Repair: Xeroderma Pigmentosum
Mottled redness and
pigmentation sign of
damage due to UV
exposure. Precursor
to cancer. 4 years old.
Carefully protected from
sunlight. 18 years old.
Works as a model.
7 Complementation Groups deficient in Excision Repair
identified by Cell-cell Hybridization.
Proofreading and Mismatch Repair
• Most DNA polymerases contain
“proofreading” activity (3’ to 5’
exonuclease); increases fidelity of
replication by 100X.
• Remaining errors fixed by Mismatch
Repair:
– How does system recognize which
strand is correct for use as template?
– In bacteria, old strands become
methylated, repair system recognizes
unmethylated new strands. Similar
system may work in eukaryotes.
Post-Replication Repair in Prokaryotes
involves an Intentional Recombination
Double-Strand Break Repair in Eukaryotes
• When both strands are broken and template can’t be
used to repair the damage, DSB repair pathway
reanneals two DNA segments.
– Homologous recombination repair uses DNA from
undamaged homologue (!)
– Nonhomologous recombination repair also occurs.
• Defects in this pathway associated with X-ray
hypersensitivity and immune deficiency.
Site-directed mutagenesis
Using mutations to study gene function in the lab
Change 1 or more nucleotides in a gene to change a triplet
codon and thus the amino acid sequence of the protein
Introduce mutated gene into animal
Determine effect on gene expression and protein function
III. Transposable Elements
• Also called Transposons or “Jumping Genes”; can move
within the genome.
• Present in all organisms; well-studied in bacteria, maize,
flies.
• Discovered in Maize Ac-Ds system:
Corn Kernel Pigmentation Phenotype
Caused by Ac-Ds Transposition:
Colored Aleurone
Barbara
McClintock,
1983 Nobel
Prize in
Medicine or
Physiology
Mendel’s wrinkled Phenotype in Peas
Also Caused by Transposon
P elements in Drosophila
• P elements are transposons in flies.
• The can be used experimentally create
mutants, mark the positions of genes, or
clone genes.
• They can also be used to insert genes
into the genome, creating a transgenic
fly.
Transposons in Humans
• Alu family of short interspersed elements (SINEs)
– Moderately repetitive DNA
– 500,000 copies of 200-300 bp repeats
• Medical example: a transposon jumped into the gene
on X chromosome responsible for hemophilia
– Not present on either X chromosome of mother
– Present on chromosome 22 of mother