Transcript Normal pairing

Mutation
The process that produces a gene or a chromosome set that
is differing than that of the wild type.
The gene or a chromosome set that results from such a
process.
Pairing between the normal (keto) forms of the bases
Mismatched bases. Rare tautomeric forms of bases result in mismatches
Transition
MUTAGENS
Chemical Agents
Radiation
Mobile Genetic Elements
Mutagens
Chemical Agents
Base analogs
Base modifying agents
Intercalators
Other classes
Millions of natural and synthetic compounds
2-AP: 2- aminopurine
Analog of adenine that can pair
with cytosine in its protonated state
Normal pairing
14
2-AP: 2- aminopurine
Analog of adenine that can pair with cytosine in its protonated state
Normal pairing
5-BU :5 bromouracil
An analogue of thymine 5-BU can be mistakenly incorporated into DNA
as a base.
The ionized form base pairs with guanine
Normal pairing
Transition
Rare form of BrU in Template
‫מוטגנים‬
Mutagens
Chemical Agents
Base analogs
Base modifying agents
Intercalators
Other classes
Millions of natural and synthetic compounds
Alkylation-induced specific mispairing.
Treatment with EMS alters the structure of guanine and thymine
and leads to mispairings
Transition
Transition
Alkelating agents
A powerful carcinogen originally isolated from peanuts infected with fungus.
Alfatoxin attaches to guanine at the N-7 position. This leads to the breakage of the
bond between the base and the sugar, thereby liberating the base and resulting in an
apurinic site. Agents that cause depurination at guanine residues should tend to induce
GC to TA transversions
Usually an A is inserted instead of the depurinated site
2-AP
EMS
NG
Mutation rate, a question of balance
Mutations
DNA repair
Mutagens
Chemical Agents
Base analogs
Base modifying agents
Intercalators
Other classes
Millions of natural and synthetic compounds
Intercalators
Flat planar molecules that mimic base pairs and are able to slip themselves in (intercalate)
between the stacked nitrogen bases at the core of the DNA double helix. In this intercalated
position, an agent can cause single-nucleotide-pair insertions or deletions
Mutagens
Chemical Agents
Radiation
Ultraviolet (UV)
Ionizing
Mobile Genetic Elements
UV light generates photoproducts
Occurs between two adjacent pyrimidines on the same DNA strand
Pyrimidine: T, C
The UV photoproducts significantly perturb the local structure of the double helix. These
lesions interfere with normal base pairing. The C to T transition is the most frequent mutation ,
but UV light also induces other base substitutions (transversions) and frameshifts, as well as
larger duplications and deletions.
Transition
Transition
Transversion
Transversion
Transversion
Transversion
MESSAGE
Mutagenes induce mutations by a variety of mechanisms. Some
mutagenes mimic normal bases and are incorporated into DNA, where
they can mispair.
Others damage bases, which then are not correctly
recognized by DNA polymerase during replication, resulting in mispairing
The Ames test
A method that uses bacteria to test whether a given chemical can cause cancer.
More formally, it is a biological assay to assess the mutagenic potential of chemical
compounds.
The test serves as a quick and convenient assay to estimate the carcinogenic potential of
a compound
Bruce Ames
1928-today
University of California,
Berkely
Ames test
Strains inactive for
BER and prone for
Entry of molecules
TA100- sensitive for reversion by
base pair substitution
TA 1535/8 frameshift
Complementation groups
Mutagenesis
Plate to select for
phenotype of interest
Complementation groups
First, we need to catalogue our mutants to complementation groups
(Total of 138 mutants were isolated in the original CTF screen).
Mutant#1
Mutant#2
Diploid
x
x
Mate
x
x
Diploid still shows
CTF phenotype
Mutant#1 and Mutant#2
are mutated in the same gene
Same complementation group
Complementation groups
Mutant#3
x
Mutant#4
x
Mate
Diploid
x
x
Diploid dont show
CTF phenotype
Mutant#3 and Mutant#4
are mutated in different genes
Different complementation groups
Complementation groups
Chromosome Transmission Fidelity (ctf) Mutants
138
Total # of mutant isolates:
101
37
19 Complementation Groups
37 Undesignated (single member)
Estimated total # of genes represented ~ 50 ctf genes
WOBBLE
A situation in which the third nucleotide
of an anticodon (at the 5’ end) can form two alignments.
This third nucleotide can form hydrogen bonds not only with its
normal complementary nucleotide in the third position but
also with different nucleotide in the position.
I stands for inosine, one of the rare bases found in tRNA, often in anticodon
MESSAGE
The genetic code is said to be degenerate because in many cases
more then one codon is assigned to a single amino acid, and, in addition,
several codons can pair with more then on anticodon (wobble)
Synthetic lethal- A screening method used to uncover mutations in a
second gene that will require the cell to maintain a wild-type copy of
the gene being studied in order to survive.
1st mutation + 2nd mutation = lethality
This screen is commonly used in yeast genetics, but can be used in
other model organisms as well.
Synthetic Lethality
yfg2
yfg1
Viable
Viable
yfg2
yfg1
Dead
Synthetic Lethality
A
aD
X
B
aD
X
X
X
bD
B
Wild-type
A
Viable
Viable
bD
Lethal
Functional Relationships
Pathway A
Pathway B
A1
B1
Complex A
Complex B
B3
A1
A2
A3
B2
B3
Essential biological function
A2
B1
A3
B2
SL interaction
B
a
Mating
b
B
a
b
A
A
Heterozygous diploid
Meiosis
B
a
B
a
b
A
b
A
Tetrad
Tetrad Dissection
BA
BA
ba
ba
Ba
Ba
bA
bA
BA
Ba
ba
bA
NPD
PDT
TT
Is this combination
lethal?
Yeast tetrad analysis (classic
method)
Step1: separate spores by micromanipulation with a glass needle
tetrad
Step2: place the four spores from each tetrad in a row on an agar plate
Step3: let the spores grow into colonies
Classical approach (tetrad dissection)
Tetrad Dissection
Tetrad
bni1∆ bnr1∆
BA
BA
ba
ba
Ba
Ba
bA
bA
NPD
PDT
BA
Ba
ba
bA
TT
We ask which mutations are synthetically lethal with our query mutant
The SGA approach allows to do it in a systematic way
Query gene
a
YFG1
yfg1::KmX
yfg2::KmX
A
yfg3::KmX
A
yfg1::KmX
YFG1
A
a
A
Mating
4700 heterozygous diploids
yfg4::KmX
A
yfg4700::KmX
A
Meiosis
Tetrads
a YFG
A YFG
a yfg
A yfg
a YFG
A yfg
a
A
YFG
yfg
a yfg
A
YFG
a
yfg
A
YFG
Selection for double mutants
yfg1::KmX
a
yfg2::KmX
a
yfg3::KmX
a
yfg4::KmX
a
yfg4700::KmX
a
Select for haploid double mutants
and score viability
yfg1::KmX
a
yfg2::KmX
a
sts1
a
yfg3::KmX
a
yfg4::KmX
a
yfg4700::KmX
a
SGA Screening Lab (University of Toronto)
Pairwise genetic interactions can be represented by a graph
8 SGA Screens:
291 Interactions
204 Genes
Genetic Interaction Network
132 Screens
4000 Interactions
1000 Genes
~200,000 Interactions/genome
Amy Tong, Fritz Roth et al., Science 303:808-813 (2004)
~2000 Quantitative SGA Screens
Yeast Genetic Interaction Network
Global Level
DNA replication
Yeast Genetic Interaction Network
Global Level
Vesiclemediated
transport
DNA replication
Yeast Genetic Interaction Network
Global Level
RNA processing
Chromatin &
transcription
Ribosome, Translation
Nuclear-cytoplasmic
transport
Mitochondria
Nuclear migration
Protein
Degradation
Peroxisomes
Chromosome
segregation and
mitosis
Vesiclemediated
transport
Amino acid
biosynthesis
DNA replication
and repair
Polarity & cell
morphogenesis
Glycosylation & cell
wall
Can we recapitulate synthetic lethality in mammalian cells?
SL interactions identified in yeast could be investigated as a candidate for novel
therapeutic target
yfg2
yfg1
yfg2
yfg2
Normal
yfg1
yfg2
Dead
yfg1
Tumor
Potential
therapeutic targets