Post on 20-Dec-2015
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
Mutagens Chemical Agents
Base analogsBase modifying agentsIntercalators Other classes
Millions of natural and synthetic compounds
2-AP: 2- aminopurine
Analog of adenine that can pairwith cytosine in its protonated state
Normal pairing14
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 DNAas a base .
The ionized form base pairs with guanine.
Normal pairing
Mutagens מוטגנים
Chemical Agents
Base analogsBase modifying agentsIntercalators
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
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 induceGC to TA transversions
Mutagens
Chemical Agents
Base analogsBase modifying agentsIntercalators
Other classes
Millions of natural and synthetic compounds
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 intercalatedposition, an agent can cause single-nucleotide-pair insertions or deletions
Intercalators
DAPI hCia2-GFP α-Tub1 MergeM
etap
has
eT
elop
has
eC
ytok
ines
is
TelophaseAnaphaseMetaphaseCIA knock-downControl
TelophaseAnaphaseMetaphase
DA
PI
α-T
ub
1M
erge
Anaphase
Telophase
Metaphase
UV light generates photoproducts
Occurs between two adjacent pyrimidines on the same DNA strand
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.
Pyrimidine: T, C
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
MESSAGE
The Ames testA 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 chemicalcompounds.
The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound
Bruce Ames1928-today
University of California,Berkely
TA100- sensitive for reversion by base pair substitutionTA 1535/8 frameshift
Ames test
Strains inactive forBER and prone forEntry of molecules
Rat liver S9 fraction is used to mimic the mammalian metabolic conditions so that the mutagenic potential
of metabolites formed by a parent molecule in the hepatic system can be assessed
Genetic screens based on random
mutagenesis have been seminal in
identifying genes involved in biological
processes such as genome stability
Yeast: A model eukaryote
Yeasts – the ultimate model eukaryote for unicellular issues and some basic cell-cell interactions
Yeast studies have broken new ground in:Cytoskeleton functions transcription mechanisms**cell cycle** transcriptional regulationorganelle biogenesis chromatin modificationsecretion* signal transductionprotein targeting mechanisms protein degradation*chromosome replication DNA repairgenome dynamics retroviral packagingprions recombination mechanismsageing function of new genesmetabolism protein modification
*Lasker Award **Nobel Prize
"for their discoveries of key regulators of the cell cycle"
Lee HartwaellPaul Nurse Tim Hunt
The Nobel Prize in Physiology or Medicine 2001
for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells
James E. Rothman Thomas C. SüdhofRandy W. Schekman
The Nobel Prize in Physiology or Medicine 2013
40
One of the major approaches that led to the understanding of cell cycle regulation was the genetic analysis of yeasts, was pioneered by Lee Hartwell and his colleagues in the early 1970s .
These investigators identified temperature-sensitive mutants that were defective in cell cycle progression. The key characteristic of these mutants (called cdc for cell division cycle mutants)was that they underwent growth arrest at specific points in the cell cycle.
cdc28 caused the cell cycle to arrest at START,
indicating that the Cdc28 protein is required for passage through this critical regulatory
point in G1
Yeast secretory pathway
Thin-section electron micrographs of SEC1 mutant cells grown at the permissive temperature (left)
and restrictive temperature (right).
The random mutagenesis approach rarely achieves saturation, because mutability, resulting in haploid viable mutants, varies widely among genes
The best way to determine the whole spectrum of genes involved in a certain phenotype
is to systematically analyze each of them
DELETION LIBRARYDELETION LIBRARY
• Availability:Availability: available commercially available commercially
• Description:Description: collection of strains deleted in all the non-essential genes collection of strains deleted in all the non-essential genes..
• Creation:Creation: replacing the gene in question by barcod containing selectable replacing the gene in question by barcod containing selectable marker based on homologus recombinationmarker based on homologus recombination
gene1::KmX
KanMX4
GENE1
gene1::KmX gene2::KmX gene3::KmX gene4::KmX gene4700::KmX
KmX is a gene that provides resistance to the drug G418
Serial analysis of deletion strains(plate based)
Apply Selection
Identify deletion strains with growth defects
1
2
3
6,000
Large-scale Mapping of Genetic and Interactions in Yeast
Synthetic Lethal Genetic Interaction in Yeast
Synthetic LethalityA
B
a
B
XA
b
Viable Lethal
a
b
Wild-type Viable
XXX
Functional Relationships
Essential biological functionSL interaction
Pathway B
A2
A3
B1
B2
B3
Pathway A
A1A1
A2
A3B1
B2
B3
Complex A Complex B
How can we detect synthetic lethal interactions between a mutant ofInterest (query gene)
and other genes
B Mating
Heterozygous diploid
Tetrad
BABAbaba
BaBabAbA
BABababA
NPD PDT TT
Tetrad Dissection
Meiosis
a b AB a
b A
B a
b A
B a
b A
Yeast tetrad analysis (classic method)
tetrad
Step1: separate spores by micromanipulation with a glass needle
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)
BABAbaba
BaBabAbA
BABababA
NPD PDT TTTetrad
Tetrad Dissection
bni1∆ bnr1∆
We ask which mutation is synthetically lethal with our query mutant
yfg1::ClonNAT
Mating
yfg1::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX
Query gene
Meiosis
4700 heterozygous diploids
Tetrads
The problem is that it is impossible to perform 4700 tetrad dissections
wt
wt wt wt wt wt
NPDPDTTT
a b
A B
a b
A B
MATa
MATα MATα
MATaa
bA
BMATa MATα
MATαMATa
a B
bA
a BMATa MATα
MATαMATabA
A B
a b
yfg1::ClonNAT
yfg1::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX
Query gene
Mating
4700 heterozygous diploidsMeiosis
Tetrads
We ask which mutations are synthetically lethal with our query mutantThe SGA approach allows to do it in a systematic way
Among the many tetrads only the MATa haploid double mutants spores can be selected
yfg1::KmX yfg2::KmX yfg3::KmX yfg4::KmX yfg4700::KmX
yfg1::ClonNAT yfg1::ClonNAT yfg1::ClonNAT yfg1::ClonNAT yfg1::ClonNAT
Use the haploid selection marker for selection
Multiplicative Model Expected Double Mutant Fitness
wt 1
0.5
0.5
Fitness
“Neutral” Expected Result, Multiplicative Model (additive)
0.25
a
b
ab
R. Mani F. Roth et. al., PNAS 2008 Mar 4;105(9):3461-6. Epub 2008 Feb 27
D. Segre, R. Kishony et al., Nature Genetics 37, 77 - 83 (2004)
S. R. Collins, N. Krogan, J. Weissman, Genome Biol 2006;7:R63. “S-Score””
Two Basic Types of Genetic Interactions
wt 1
0.5
0.5
“Negative” Synthetic Lethal
“ Neutral”Expected Result
a
b
ab
Fitness (colony size)
1
Two Basic Types of Genetic Interactions
wt 1
0.5
0.5
“ Neutral”Expected Result
0.5
“Positive”e.g. two genes whose products are inthe same nonessential complex/pathwayEpistasis
a
b
ab
FitnessFitness (colony size)
0.5
0.5
0.5
Proteincomplex
A
B C
Non-functionalB C
Non-functionalA
C
Non-functionalC
Synthetic lethal interactions
Conditional alleles of sec13 and sec23 show synthetic lethality at low temperatures
Genes that act in the same pathway (or complex) will share the same synthetic lethal interactions
A complete map of all possible genetic interactions has the potential to reveal all pathways and complexes!
Pairwise genetic interactions can be represented by a graph
8 SGA Screens:291 Interactions204 Genes
8 SGA Screens:291 Interactions204 Genes
Genetic Interaction Network132 Screens
4000 Interactions1000 Genes
~200,000 Interactions/genome
Amy Tong, Fritz Roth et al., Science 303:808-813 (2004)
•All dynein subunits are SL with the same subset of mutants•YMR299c is probably a new component of the dynein complex
dyn
ein
SGA analysis clusters related genes
Array gene clusters
Qu
ery
gen
e cl
ust
ers
Ribosome, Translation
Mitochondria
Vesicle-mediated transport
Glycosylation & cell wall
Polarity & cell morphogenesis
DNA replication and repair
Chromosome segregation and
mitosis
Nuclear-cytoplasmic transport
Chromatin & transcription
Peroxisomes
Amino acid biosynthesis
RNA processing
Nuclear migrationProtein
Degradation
Yeast Genetic Interaction NetworkGlobal Level
yfg1
yfg2
Dead
yfg2yfg1
yfg2
yfg2
Normal
Tumoryfg1
yfg1
yfg2
SL interactions identified in yeast could be investigated as a candidate for novel therapeutic target
Blue circle, SiRNA targeting central gene alone
Red circle, SiRNA targeting cancer gene alone
Yellow triangles, predicted viability of double siRNA treatment
Green circles, observed viability of double siRNA treatment
Solid grey line, interaction observed in bothS. cerevisiae and HCT116 cells.
Green dashed line, interaction observed only in S. cerevisiae.
Orange dotted line, interaction observed only in HCT116 cells.
Mammalian genetic interaction network