Download Recombinant DNA II

Announcements
1. Lab reports: as printed in the X-linked cross lab write-up, you are
expected to perform chi-square analysis on your data (both F1 and
F2 for each cross - total of 4 chi-square tests); this will be basis for
your discussion, ie. was there significant deviation between expected
and observed ratios? New deadline: Fri. Nov. 8th at start of lecture.
2. Problem set 5 is graded and in folders in lab; avg. was 12/15. We’ll
go over correct answers in lab this week; we’ll also take questions re.
prob. set 6.
3. Ch. 17 rdg: skim pp.456-458; 460-461;
Review of Last Lecture
1. Eukaryotic Gene Expression:
it’s more complicated being multicellular
2. The Promoter
3. Enhancers
4. Methylation
5. Alternative splicing
Outline of Lecture 28
I. Post-transcriptional gene regulations:
Alternative splicing
II. Classification of mutations
III. Detection of mutations in humans
IV. Different forms of mutations
I. Post transcriptional gene regulation
If humans have approximately the same number of
genes as a fruit fly, and we require more complex
cellular functions (presumably with a larger number
of proteins) - how do we accomplish this?
Making different forms of a protein from a single gene
Differential splicing of exons
Processing Control: Differential
Splicing of IgM genes
Alternative splicing
1. Chromosomal ratio
activates txn of Sxl in
females only
2. SXL controls splicing of
tra-2 mRNA
3. Females: exon 2 (which
has a stop codon) is
removed via SXL
Males: exon 2 is not
removed.
4. Males: no active TRA
Females: TRA is made.
5. TRA directs splicing of dsx
mRNA in specific
manner; in males default
splicing occurs.
II. Classification of mutations
A. Spontaneous vs Induced
B. Gametic vs Somatic
C. Phenotypic Effect:
– Morphological, e.g. pea, fly traits
– Biochemical, e.g. hemophilia, leu– Behavioral, e.g. mating behavior in flies
– Regulatory, e.g. in lac operon; important for evolution
– Lethal, e.g. Huntington disease, Manx allele
– Conditional, e.g. temperature-sensitive allele
III. Detection of mutations in humans
- Pedigrees
- Dominant mutations easiest to detect
Where did original mutation occur?
Could it be an X-linked mutation?
Gamete of parent - gen.I
Probably not
What is the Rate of Spontaneous Mutation?
• Bacteriophage T2: 10-6 to 10-8 per gene replication.
• E. coli: 10-5 to 10-10 per cell division.
• Maize: 10-5 to 10-6 per gamete per generation.
• Drosophila: 10-5 to 10-6 per gamete per generation.
• Human: 10-5 to 10-6 per gamete per generation.
Neutral vs. deleterious mutations
Most spontaneous mutations will occur in 95% of genome that
does not encode genes - neutral mutations.
What is rate of deleterious mutations in humans?
1.6 deleterious genetic changes/individual/generation
Consequences to our species? Read short article by
James Crow on page 465-466 in text.
IV. Different forms and origins of Mutations
More types of mutations
•Normal
THE ONE BIG FLY HAD ONE RED EYE
•Missense
THQ ONE BIG FLY HAD ONE RED EYE
•Nonsense
THE ONE BIG ***
•Frameshift
THE ONE QBI GFL YHA DON ERE DEY
•Deletion
THE ONE BIG HAD ONE RED EYE
•Insertion
THE ONE BIG WET FLY HAD ONE RED EYE
•Duplication
THE ONE BIG FLY FLY HAD ONE RED EYE
•Expanding Mutation
–Generation 1
THE ONE BIG FLY HAD ONE RED EYE
–Generation 2
THE ONE BIG FLY FLY HAD ONE RED EYE
–Generation 3
THE ONE BIG FLY FLY FLY HAD ONE RED EYE
Fragile X Syndrome: the Result of an
Expanding Mutation Over Time
•
Mental retardation, characteristic facial
structure.
•
CGG is present in 5’ UTR of FMR-1
gene:
– wildtype: 6-54 repeats
– carrier: 55-200 repeats
– affected: > 200 repeats
•
> 200 repeats, DNA becomes
methylated, inactivating the gene.
•
Function of protein still unclear.
•
Other examples: one form of muscular
dystrophy, Huntington’s disease.
Different forms 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)
1. Tautomeric Shifts Cause
Spontaneous Mutations
• Can’t be avoided - inherent in the chemistry of bases.
• Always a transition: changing one purine (G or A) to
the other purine, or one pyrimidine (C or T) to the
other pyrimidine.
• DNA proofreading minimizes the mutation rate due to
tautomeric shifts. This involves 3’-5’ exonuclease
activity in DNA polymerase.
Keto-Enol Tautomeric Shifts in T and G
common
rare
Amino-imino Tautomeric Shifts in C and A
common
rare
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 WWI.
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 -> T transition (and G -> A transition)
(b) Causes: A -> G transition (and T -> C transition). All.
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).