Transcript 19 Dominant Negative Examples

1
GENETICS IS A STUDY OF HOW PROTEINS INTERACT, FOLD, AND FUNCTION
While classical genetics often concerned itself with the rules governing the
segregation of genetic material, modern genetics is more concerned with
uncovering the function of proteins (mainly) and the elucidation of pathway
organization.
The next few lectures will stress how defects in genes lead to discrete
alterations in the functions of proteins. Through these lectures we hope
to provide you with an appreciation of the molecular defects caused by
particular types of mutations in genes and how they result in the phenotypes
observed in the organism.
We will also discuss how one can use mutations in a given gene to identify
more genes that function in a given pathway and how to organize a genetic
pathway.
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Classes of mutants
Recessive
Loss of function, often premature termination of a polypeptide
Dominant
1) Hyperactivity (Deregulated activity)
A) Mutation in negative regulatory domains
B) Increases function (better catalyst, stronger interaction)
2) Increased expression (Usually cis acting sequence point mutants or rearrangements)
A) Mutation of negative regulatory sequences or introduction of new positive
elements in cis to a structural gene (includes heterochronic and ectopic expression)
B) Gene amplification
C) Mutations that increase RNA or protein stability
3) Novel function
A) Suppressor tRNAs (INFORMATION SUPPRESSORS)
B) Altered specificity of a DNA binding protein
C) Generation of a new regulatory sequence in front of a gene
D) Altered enzymatic activity
E) Mislocalization
4) Dominant negative
A) Subunit mixing
B) Blocking processive processes (drug sensitivities)
5) Haploinsufficiency
(Can see more often in a sensitized background)
3 Classes of mutants
Recessive -Loss of function, often premature termination of a polypeptide
Many types of mutations can result in a recessive phenotype.
Null alleles:
1) Deletion
2) Premature stop codons, preferably early in the protein.
-Truncated proteins often contain partial activities.
Partial loss of function alleles: (a.k.a. hypomorphs)
Altered activity
These alleles can result from partial inactivation of function such as mutating the
site for a binding protein such that a lower concentration of the complex between
the two proteins exists. Another example is mutations in the active site of an enzyme
resulting in a reduced enzymatic activity.
Altered abundance
1.
2.
3.
Mutations can alter the stability of a protein to reduce its levels.
Mutations can reduce the level of expression by removing positively acting
sequences controlling transcription, mRNA stability, or translation.
Decreasing the efficiency of folding can result in a mixture of correctly and
incorrectly folded proteins.
Altered localization
Subcellular localization is an important aspect of protein function. Mutations in
targeting sequences cause loss of function.
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Levels at which mutations can disrupt protein function.
Coding Region
Enhancer
Transcription
+ Processing
mRNA
AUG
AAAAA
Translation
Regulation
Factors governing
the overall function
of a protein
-Abundance
-Location
-Activity
Translation
mRNA Abundance
Translation
Folding
Stability
Localization
Activity
5 Classes of mutants
Dominant
Gain of function.
1) Hyperactivity
A) Increased function
better catalyst, stronger interaction
B) Mutation in negative regulatory domains
genetic switches stuck in the ON or OFF position
Proteins are machines that can be turned on and off. Many of the more interesting
mutations in nature affect these regulatory switches. While the vast majority of
mutations we deal with as geneticists are recessive, the rare dominant mutations
are often the most interesting. These are often in signaling molecules or key regulators.
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Hyperactivity
A) Increased function
Increasing the activity of a protein could be accomplished by changing its specific activity.
i.e. reduce the activation energy, G‡, of an enzymatic reaction.
Increasing the binding affinity of one molecule for another.
For example imagine a situation in which a DNA binding protein is activated by a signal to
bind a particular piece of DNA. In the absence of the signal, the DNA binding protein has a
low affinity for the sequence in question that together with its concentration prevents high
occupancy of the cognate sequence on the chromosome.
DBP + DNA
Keq =
DBP-DNA Complex
[DBP-DNA complex]
[DBP] [DNA]
The signal activating the DNA binding protein does so by increasing the equilibrium
constant, by increasing the affinity of the DNA binding protein for the DNA.
This same outcome can be accomplished through mutations that:
1) Increasing the concentration of the protein
2) Increasing the affinity of the protein for the cognate DNA site in the absence of signal.
Depending upon how the protein works, an increased occupancy for the DNA could
be accomplished by increasing the form of the protein that binds DNA if for example a dimer
was needed. It could also be accomplished by increasing the number of contacts or strength
of existing contacts the DNA binding protein makes with the DNA sequence to which it is bound.
7 Dominant uninducible mutations in the bacterial trpR gene
TrpR is a homodimeric DNA binding protein that requires tryptophan for binding to DNA
and repression of tryptophan biosynthetic enzymes.
Tryptophan
TrpR
Trp biosynthetic genes
Operator Site
When tryptophan levels are low, there is insufficient tryptophan to bind TrpR and stablize the homodimers
association with DNA. This reduces the occupancy of promoters and results in transcriptional induction.
The binding affinity for the repressor on DNA is determined by the energy derived from the sum of three interations:
1) Tryptophan-TrpR interactions,
2) TrpR-TrpR interactions, and
3) TrpR-DNA interactions.
G binding = G1 + G2 + G3
In a screen for altered specificity trpR mutants, a mutant was discovered that allowed TrpR to bind to mutant
operator sites. This mutant was also uninducible when bound to its native site, i.e. it bound in the absence of
tryptophan.
Why?
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In response to DNA damage, ssDNA is produced, RecA is activated and together
with residues on LexA catalyzes the autoproteolysis of LexA.
This results in loss of LexA dimerization and DNA binding activity and causes
transcriptional induction.
Changes of amino acids on LexA that are involved in either recognition by RecA
or in the proteolysis mechanism itself result in uninducible, dominant phenotypes.
Question:
Why are these mutations dominant?
Under what circumstances might they be recessive?
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Dominant constitutively acting mutants of Protein kinase C
Protein kinase C (PKC)is a protein kinase that acts as the recipient of
diacyl gylcerol (DAG) and Ca++ signals
Kinase Domain
Inhibitory Domain
PKC
Pseudosubstrate Site
DAG
Active Site
Ca++
DAG Ca++
Mutants causing truncation of the inhibitory domain of PKC remove the inhibitory
psuedosubstrate site and activate the kinase just as if DAG and calcium were present.
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Dominant constitutively acting mutants of Cdc2
Cdc2 is a protein kinase that regulates cell cycle transistions. It is positively regulated by binding
to cyclins and negatively regulated by phosphorylation of tyrosine 15 in its ATP binding site.
The timing of phosphotyrosine removal regulates entry into mitosis. Premature activation
of Cdc2 causes premature entry into mitosis.
Wee1(tyrosine kinase)
Perpendiculars indicate
inhibitory signals
Cdc2/Cyclin B
Arrows indicate
stimulatory signals
Cdc25 (tyrosine phosphatase)
Mutants in which the phosphorylation target tyrosine 15 is mutated to phenylalanine
create a Cdc2 mutant that is resistant to the inhibitory actions of Wee1 and
is therefore hyperactive causing premature mitotic entry.
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Hyperactive mutants in the GTPase Ras
Ras is a small GDP and GTP binding protein that acts as a molecular switch. In its GDP bound
form, it is considered to be "OFF". In its GTP bound form it is considered to be "ON".
Dominant acting mutations in ras are responsible for a large percentage of human cancers.
It is important to understand the switch in order to learn how to turn it off.
Ras is positively regulated by exchange factors, GEFs, that convert the GDP bound form into
a GTP bound form.
Acting in opposition to GEFs are GAPs, which are proteins that activate an intrinsic
GTPase function on Ras.
Activating signals
GEF
OFF
Ras( GDP )
Ras( GTP )
ON
GAP
Signals
Mutations that affect the equilibrium between the GDP bound state and the GTP bound state can
have phenotypic consequences. The Ras mutation val19 in which amino acid 19 is changed to valine
severely reduces the intrinsic GTPase activity of Ras. As a result, Ras accumulates in the GTP-bound
state and is active without upstream signals.
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Dominant Mutants -Consitutive Expression
Constitutive expression usually results from cis acting mutations that remove inhibitory
sequences or create new stimulatory sequences in processes that are rate limiting for
production of a protein.
Although at first glance it seems there should be only one rate limiting step in a
given process, the production of a protein is comprised of many distinct steps each
of which have rate limiting components that can in a multiplicitive way influence the
eventual rate of the process.
Ectopic and Heterochronic expression.
Ectopic expression is the expression of a protein in a cell in which it is not normally
expressed. This applies primarily to multicellular organisms (Metazoans).
Heterochronic expression is the expression of a protein at the incorrect time, such as
earlier than it should normally be. This applies to cell cycle regulated proteins that
are periodically expressed and to developmentally regulated proteins.
Inappropriate expression can have dominant phenotypic consequences.
Why?
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Increased Abundance
Mechanisms to enhance the abundance of a protein.
Silencer Enhancer
Coding Region
1) Remove silencing elements (repressors, imprinting)
2) Add additional enhancers
Transcription
+ Processing
mRNA
AUG
AAAAA
Translation
Regulation
Translation
3) Enhance transcription rate (loss of pausing sites)
4) Increase mRNA stability (i.e. abundance)
5) Enhance splicing of the differential splice of interest
6) Enhance protein stability (removal of instability elements)
7) Enhance proper localization (competition)
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Dominant Mutants
-Novel function (Neomorphs)
These mutations alter the protein such that it has a novel function. Classically
a dominant mutant whose phenotye is not altered by the addition or subtraction of wild-type
copies of the gene can be considered to be a neomorph, although you can see that this
definition is somewhat lacking in clarity.
For example ectopic and heterochronic mutations fall into this definition. In fact
often a particular mutant will fall into more than one category, so do not be alarmed when
you discover this.
Examples
1)Information suppressors.
tRNA suppressors that now recognizes a stop codon and instead of terminating
the polypeptide, it inserts an amino acid.
2) Altered specificity of a DNA binding protein.
Mutations in the DNA recognition domain of a DNA binding protein that now allow it to
recognize a novel DNA sequence, thereby placing new genes under its regulation.
This can be broadened specificity as well.
3) Altered specificity of protein-protein interactions. There are many examples of members
of protein families that bind in a specific way to members of other protein families,
for example leucine zipper motifs. Mutations can potentially alter the specificity of
such interactions leading to novel partner formation and dominant effects.
4) Altered enzymatic specificity. Example evolved -galactosidase, ebvA. This is an enzyme
that normally hydrolyzes a complex sugar other than lactose. However, by mutation its
specificity is altered to now include lactose.
15 Dominant Negative Mutations
Dominant negative mutations are mutant forms of proteins that
interfere with the function of the wild-type proteins.
---hypomorphic or a null phenotypes.
This class of mutants is often used to explore protein function in systems
with poor genetics.
It is primarily based on the idea that proteins work by interacting
with other proteins, a titratable property.
Dominant negative is often improperly used.
Criteria
1) Phenotype must resemble the hypomorphic phenotype
2) Additional copies of WT protein should lessen the phenotype.
16 Dominant Negative Examples
Subunit mixing.
A tetrameric protein in which all four subunits must be wild-type in order for the
protein to function.
If half of all monomers are mutant, then only 1/16th of the resulting tetramers will
contain no mutant proteins and will function as wild-type.
Does this mean the organism will have a mutant phenotype?
Phenotypic threshold.
Given the fact that equal levels of the WT and mutant protein are produced, is it
necessarily the case that only 1/16th of the wild-type activity will result?
Ribonuclotide reductase and feedback circuits.
17 Dominant Negative Examples
Transcription factors often have separable
activation and DNA binding domains.
ACT
DBD
Expression of a mutant lacking the
activation domain can compete for binding
DBD
Overproduction of the deletion mutant is a
strategy employed to enhance the interference
and achieve the phenotypic threshold .
18 Dominant Negative Examples
Blocking processive proceses.
DNA synthesis
RNA synthesis
Protein synthesis
R
R
R
S
Inhibitor
R
R
Drug resistance is recessive.
Drug sensitivity is dominant.
S
19 Dominant Negative Examples
Topoisomerases.
In the course of altering the superhelicity of DNA, topoisomerases bind
particular sites and cleave DNA and remain covalently bound to the DNA, allow
a topological event to take place, and then religate the DNA.
Various drugs can block different steps in this process.
Some drugs may prevent binding to DNA.
Others may block the religation step, causing the topoisomerases to remain bound
to their site of action.
Mutant topoisomerases exist that do not bind the drug in question and are
therefore resistant to inhibition.
Would you expect these drug-resistant mutants to be dominant or recessive?
19 Dominant Negative Examples
Topo I
Drug A
Drug B
Drug resistant mutants: Dominant or Recessive?
20 Dominant Negative Examples
Heterotrimeric complex ABC
A
C
B
Overproduction of Wild-type Protein B
B
A
B
B
B
C
B
This is an example in which overproduction of a wild-type
protein interferes with its own function.
21 Haploinsufficiency
Unusual case in which a null mutant causes a phenotype in the presence of a
wild-type allele of the gene.
CRT1
A repressor of transcription whose levels are very tightly controlled.
Loss of one copy give a partial derepression of transcription units under its regulation.
Haploinsufficiency occurs very rarely and only proteins whose levels are
very critical give this phenotype. The mutation must be a null to qualify.
In developmental pathways, proteins that provide quantitative effects may
be prone to haploinsufficiency. i.e. morphogen gradient proteins.
Other cases of gene classes that might be prone to haploinsufficiency are
proteins that form higher order structures such as tetramers A+A+A+A = (A)4.
If the binding constant is near the concentration of A in the cell, then the
concentration of A to the fourth power governs the concentration of the tetramer.
Haploinsufficiency is actually much more easy to detect in pathways that have
already been sensitized by mutations that already produce a mild phenotype.
This observation is the basis for one of the most useful screens employed
by Drosophila geneticists.
22 Conditional Mutations
What does a conditional phenotype mean at a molecular level?
Types, Ts, Cs, Salt-sensitive, degron
Are all ts mutations the same?
Different types of ts mutants
1) ts-synthesis.
2) ts-activity.
3) ts-stability.
4) ts-process.
23 Different types of ts mutants
1. Temperature sensitive synthesis
In this type of mutant, proteins made at the permissive temperature are
temperature resistant. Only newly made proteins are inactive.
This is generally interpreted to mean that the process of protein folding is
temperature sensitive. Once this type of mutant protein is folded at the
permissive temperature, it is as stable as the wild type protein and has full activity.
Implications
in vitro
Protein
Activity
in vivo
0 1
2 3 4
Time after temperature shift
24 Different types of Ts mutants
2. Temperature sensitive activity
In this type of mutant the activity of the protein is Ts regardless of
whether it was synthesized at the permissive or non-permissive
temperature. These mutants are also Ts in vitro.
Implications
Protein
Activity
in vitro
and
in vivo
0 1
2 3 4
Time after temperature shift
25 Different types of Ts mutants
3. Temperature sensitive stability
It is thought that this type of mutant becomes partially unfolded
at the non-permissive temperature and therefore accessible to the
cells protease machinery. This class of mutants are unstable in vivo
but retain in vitro activity when purified.
Implications
in vitro
Protein
Activity
in vivo
0 1
2 3 4
Time after temperature shift
26 Different types of Ts mutants
4. Temperature sensitive process
This class of mutants are actually null mutations * that result in a temperature
sensitive phenotype.
*Does not have to be a null, but those are the easiest to definitively demonstrate.
Examples
1.
SPA1 spindle pole antigen
-Bridging protein stabilizing a structure
2.
SSN6 - general repressor of gene expression.
-Derepression of lots of genes results in pleitropic phenotypes and Ts.
3.
The heat shock response.
Mutations in genes that are involved in dealing with changes in
temperature might produce a temperature sensitive phenotype, even if their
activities are not affected by temperature.
Few Ts mutant proteins fall neatly into a single category with the exception of
the Ts process. Often a Ts stability mutant will also have some defect in activity
as well, etc.
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REMEMBER, when you raise the temperature, you are not only
affecting the activity of your mutant protein, but also the physiological
state of the cell and organism, therefore 2 variables are simultaneously
varied. The control becomes critical.
What are the advantages of using ts mutations?
There are many advantages, especially for essential genes.
"Death is not a very interesting phenotype"
1) They allow us to easily work with mutations in essential genes in a haploid organism.
2) They allow us to observe changes in the organism as we change from permissive
to non-permissive conditions.
3) They allow simple isolation of suppressors which may themselves be involved in
the same process. More on this later.
4) They can be useful for determining the order of function of genes in a pathway.
28 Suppressor or Pseudoreversion Analysis
Suppressors are mutations that partially or totally restore a particular phenotype to
a mutant strain.
Suppressor analysis is an attempt to use a mutation in a gene to identify other genes
relevant to a particular process to make us better equipped to solve the underlying
biochemical basis of the process.
True Revertants vs Suppressors
1. True revertants are mutations that reverse the original mutation back to the
wild-type amino acid.
2. Intragenic revertants can be true revertants or secondary mutations in the same
gene that restore function to the protein.
1) If a mutant protein is non-functional because it now lacks a particular amino acid
that held an alpha helix in place, this mutation can be compensated for by a second
mutation that now restore the position of the helix.
2) If a mutant protein is non-functional because it has Ts-stability, a secondary mutation
that overproduces the mutant protein might be able to restore enough of the protein to
restore an apparent wild type phenotype.
Second site suppressors are mutations in different genes, usually genetically unlinked,
that compensate for the defect of the original mutation.
29 Suppressors of a pab1 Ts mutant
Alan Sachs had a Ts mutation in the yeast PAB1 gene which encodes the polyA
binding protein which binds to the polyA tail of eukaryotic mRNA. The Ts mutant
failed to grow at 37°C.
He selected for spontaneous revertants capable of growing at 37°C. Among these
he chose to study those with a Cs phenotype.
25% were in the pab1 Ts gene itself.
10% were dependent upon the pab1 Ts mutation for their Cs phenotype.
65% were able to suppress a pab1 deletion.
Why Cs mutations?
Why spontaneous mutations.
30 Allele-specific suppressors
What is an allele specific suppressor in molecular terms?
1. Two functions in one gene. Some mutants may be defective for one function,
another might be defective for both. If the suppressor was isolated for the mutant
with a single defect, it may be incapable of suppressing the mutant defective in both.
2. Restoration of specific protein-protein interactions.
+_
A
B
_
+
Wild-type
interaction
__
A1 _ B
+
+_
A2 _ _ B
Mutants A1 and A2 have
weaker interactions with B
Suppressors can restore interactions
_+
++
A1 _ B1
A2 _ _ B1
+
The B1 allele can suppress A1 but not A2 mutants.
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Allele-specific suppressors
3. Degree suppressors in an allelic series.
90% in pathway A
10% in pathway B.
Need 50% activity to survive or score as a suppressor.
100%
75%
Activity
A5(60%)
50%
A4(35%)
25%
A3(25%)
A2(10%)
A1(5%)
Allele-specific suppressors of A mutations in pathway B.
Allele
%Activity
Suppresses
B1
B2
B3
B4
B5
20%
30%
40%
45%
60%
A4
A4, A3
A4, A3, A2
A4, A3, A2, A1
A null
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Allele-specific suppressors
In the pab1 Ts suppressor screen, one class of suppressors was absent
-the allele-specific suppressors that retain their Cs phenotype in the presence
-of wild-type PAB1.
Why?
1) Impossible.
2) Rare.
3) Window of selection.
Yeast were grown at 25°C prior to selection at 37°C.
Would you expect a suppressor of a Ts mutation to be Cs?
Do you expect to miss classes of suppressors by choosing only Cs suppressors?
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Suppressors
Are suppressors usually in the same pathway as the mutation they suppress?
How does one know?
If the suppressor alone, or a different mutation in it, has a phenotype
similar to the mutation it suppresses.
Overproduction Suppressors.
Gene dosage suppression is very popular in yeast molecular genetics. Why.
Examples.
Cdc28 isolated Clns and Cks1 as suppressors.
How can overproduction of a protein suppress a mutant?
A+B
AB
Forcing and equilibrium to the right and increasing AB.
In principle it is similar to suppression by point mutations.
34 Other methods for identification of genes in a given
pathway using existing genes or mutants.
Cloning by association.
Two hybrid systems
OFF
A
His UAS
HIS3
G
Activation
Dom ain
A
B
ON
His
UAS
G
HIS3
+
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Unlinked non-complementors
Mutations in an unlinked gene that fail to complement the original mutation.
Uppercase = wildtype, lowercase = mutant
A/A B/B = wt, A/a B/B =wt,
A/a B/b = mutant phenotype
This has been used as the basis of screens to identify interacting genes.
Worked well for tubulin.
How does it work?
1) Phenotypic threshold.
2) Poisoned link of a chain.
Screens are performed by mutagenizing a population and mating it
to an organism containing a mutation in a particular gene. Screen
for lack of complementation. Screen those for absence of linkage.
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Enhancer and Suppressor screens.
This type of screen in similar in principle to the synthetic lethal screen, however it
is usually performed in a diploid
This screen starts with a mutant background that has a detectable phenotype.
a/a X/X x (a/a x/X)*
* indicates mutagenized males
produces a doubly heterozygous animal a/a b/B
where the small b is an allele generated by mutagenesis
The "b" allele could be a null allele or a dominant mutation.
This screen can detect enhancers or suppressors that alter the initial mutant phenotype
and is very commonly used in Drosophila mutant hunts because it can be performed in
a single generation without the need to back cross the mutants to make them
homozygous. This saves significant amounts of time and allows many more
organisms to be examined, i.e. rarer mutants to be discovered.
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Synthetic phenotypes.
When a combination of mutations produces a phenotype more severe than either of
the mutations alone.
Synthetic lethality is often used in haploid organisms.
Yeast sectoring screens for synthetic lethal mutants.
This screen is performed on haploid yeast.
Colony
Red Color
Plasmid
URA3
Mutant
Sector (WHITE)
Red Sectoring
GENE X
Wild-type
Sector (RED)
Mutant in Gene X(Ts)
Non-sectoring
Chromosome
Screen for mutagenized colonies that fail to lose the color marked plasmid containing the
wild-type gene.
A percentage of such colonies will contain second mutations that in combination with the
initial mutation are lethal.
38 Pathway Organization - How do you know if two genes are in the
same pathway or in a different pathway that affects your phenotype?
Radiation sensitivity. Many different pathways contribute to repair of DNA damage.
Epistasis Groups
RAD52 Group
Recombinational
Repair
RAD9 Group
Cell Cycle
Coordination
RAD 3 Group
Excision Repair
Combining mutations in genes in different groups should cause synergystic sensitivities.
Double mutants among genes in the same pathway generally have the same phenotype are the
mutations in each gene alone.
Exceptions.
Some genes are in more than one pathway.
39
Gene Order (Double Mutant Analysis)
EPISTASIS Describes the relationship between genes.
Epistasis Groups = Pathways
Epistatic interactions between genes is used to define gene order.
Definition of Epistasis: When two genes of differing phenotypes are
combined together in one organism, the epistatic gene is the gene
whose phenotype dominates.
Interpretation:
This depends on what phenotype you are considering.
If you are looking at accumulation of an intermediate in a pathway
or an unusual structure unique to a particular phenotype,
then the epistatic gene is upstream.
40 How does it depend on the way you are looking at the system?
Good examples of this are the adenine biosynthetic pathway and the cell division cycle genes.
If you are looking at a downstream event in a regulatory pathway, such as transcription,
the epistatic gene is downstream.
Key to interpretation:
Use common sense.
Pathway Organization
Two mutants with similar phenotypes same pathway or different?
Rule of thumb - if the double mutant has the same phenotype as the single mutations alone,
the two genes are thought to be in the same pathway.
What about synthetic phenotypes? Don't we use double mutants that enhance each others
phenotypes to identify genes in the same pathway? What gives?
It depends on the kind of mutations you are using.
41
Gene Order
How can we use genetics to reveal the organization of a genetic pathway?
Double mutant combinations
The GAL1 gene is inducible in the presence of galactose
gal4 mutants fail to induce GAL1 mRNA in response to galactose
gal80 mutants constitutively express GAL1 without galactose
Possible orders of function.
AB
BA
GAL4
GAL80
GAL80
GAL4
GAL1
GAL1
Separate Pathways
GAL4
GAL1
gal80 gal4 double mutants are uninducible.
Which pathway is correct?
GAL80
42 Pathway Organization
Biosynthetic Pathways
Example: Hypothetical tryptophan biosynthetic pathway. Intermediate feeding experiments.
C 1, C 2, and C3 are biosynthetic intermediates
tryptophan
C1
C3
C2
X
Observations
1. Mutants in trpA can grow when supplied with C 2 and C 3 but not C1
2. Mutants in trpB can grow when suppplied with C1, C2, and C3
3. Mutants in trpC cannot grow when with any of these intermediates
4. Mutants in trpD can grow with C3.
5. Double mutants between trpB and trpD can grow with C3.
D is said to be epistatic to B
What are the order of function of the genes?
There are differences between regulatory and biosynthetic pathways concerning the use
of the word epistasis.
The epistatic mutation is the one whose phenotype is observed in the double mutant.
However, depending upon which property is being measure, the epistatic gene can be
upstream or downstream in the pathway.
43 Adenine Biosynthesis and Epistasis
C1
ADE3
ADE2
C2
Red
ade3 mutants are white,
ade2 mutants are red,
ade2ade3 double mutants are white
Adenine
ADE3 is epistatic to ADE2
ade3 mutants grow on C2 or adenine,
ade2 mutants grow only on adenine,
ade2ade3 double mutants grow only on adenine. ADE2 is epistatic to ADE3
The epistatic mutation is the one whose phenotype is observed in the double mutant.
However, depending upon which property is being measure, the epistatic gene can be
upstream or downstream in the pathway. If you look at the red color, ade3 is epistatic
to ade2 mutations, if you look at feeding intermediates, ade2 is epistatic to ade3,
but the order of the pathway is the same in both.
44
Pathway Organization
Morphological Pathways
Example: Phage T4
Reciprical shift experiments.
This allows the use of different types of conditional mutants to independently
manipulate the functions of 2 genes.
Ts and Cs mutations in Phage T4 morphogenesis
Gene 1 Ts
Gene 2 Cs
Gene 2 Cs
Gene 1 Ts
T4
T4
(NPT for Gene 1)
(NPT for Gene 2)
18°C
(NPT for Gene 1)
37°C
(NPT for Gene 2)
37°C
18°C
45 Circular Pathways - The Cell Cycle
Using different types of conditional mutations and morphological markers to determine
the order of gene function.
F
START
G1
cdc15
Noc
cdc28
HU
M
S
rnr1
G2
cdc13
If cdc mutants arrest with different phenotypes, i.e. G1 vs G2, they can be ordered on the
basis of morphological markers.
Many different cdc Ts mutants arrest with large buds and undivided nuclei.
How can they be ordered?
Relative to a third marker such as DNA replication. Example cdc13 and rnr1.
46
The Power of Selection
Yeast as an example.
14 Mb genome
1.4 x 107 base pairs x 3 = 4.2 x 107 possible single base changes.
This does not represent every possible AA change.
UAX = Tyr
CCX = Pro
UCX = Ser
The genetic code presents
constraints on evolution
CUX = Leu
Spontaneous Mutation
10-9 per base/generation
1 Kb gene 10-6/generation
A null allele in a given gene occurs spontaneously with a frequency of 10-5 to 10-6/generation.
lacI
STOPS
90% of all positions can tolerate any amino acid
10% can tolerate only a few different amino acids
Chemical mutagenesis can increase mutation rates several orders of magnitude.
Each chemical causes a different spectrum of base pair changes,
a reflection of chemistry and repair systems.
47
46
-Factor (mating pheromone)
F receptor
STE2
 
GPA1
gpa1 mutants ON
ste mutants OFF

STE4 STE18
STE20 (kinase)
STE7, 11 (kinase)
FUS3, KSS1 (kinase)
STE12 (TF)
Fus1-lacZ Reporter
4847
-Factor (mating pheromone)
F receptor
STE2
 
GPA1
gpa1 mutants ON
ste mutants OFF
gpa1 ste2 ON
gpa1 ste4 OFF
gpa1 ste18 OFF
gpa1 ste X OFF

STE4 STE18
STE20 (kinase)
STE7, 11 (kinase)
FUS3, KSS1 (kinase)
STE12 (TF)
Fus1-lacZ Reporter
4948
-Factor (mating pheromone)
F receptor
STE2
 
GPA1
Dominant mutants
Activating alleles of
STE20
STE7
STE11

STE4 STE18
STE20 (kinase)
STE7, 11 (kinase)
FUS3, KSS1 (kinase)
STE20* ste2 ON
STE20* ste4 ON
STE20* ste18 ON
STE12 (TF)
STE20* ste7 OFF
STE20* ste11 OFF Fus1-lacZ Reporter
STE20* ste12 OFF
50
49
-Factor (mating pheromone)
F receptor
STE2
 
GPA1
Dominant mutants
Activating alleles of
STE20
STE7
STE11
STE7* ste2
STE7* ste4
STE7* ste18
STE7* ste20
STE7* ste11
STE7* ste12

STE4 STE18
STE20 (kinase)
STE7, 11 (kinase)
FUS3, KSS1 (kinase)
ON
ON
ON
STE12 (TF)
ON
OFF Fus1-lacZ Reporter
OFF
50
51
-Factor (mating pheromone)
F receptor
STE2
 
GPA1
Dominant mutants
Activating alleles of
STE20
STE7
STE11

STE4 STE18
STE20 (kinase)
STE7, 11 (kinase)
FUS3, KSS1 (kinase)
STE11* ste2 ON
STE11* ste4 ON
STE11* ste18 ON
STE12 (TF)
STE11* ste20 ON
STE11* ste7 OFF Fus1-lacZ Reporter
STE11* ste12 OFF
One can also use biochemical events to order
gene function in pathways. For example,
phosphorylation of proteins can be used.
52
Genetic Screens in Yeast
1) Can you devise a selection or easy screen?
2) How tight?
3) Secondary criteria are important.
4) Mutagenize 2 haploid strains of opposite mating type with
different selectable markers for future diploid selection
Strain 1
MATa trp1
Strain 2
MAT his3
5) Select mutants
6) Test dominance/recessiveness
7) Place into complementation groups by mating
8) For dominant mutants test allelism by linkage
9) Organize pathway by epistasis analysis
10) Clone genes by complementation, etc.
A + 
A/
Diploid
Sporulation
Haploid
53
52
Genetic screens in yeast 2
A screen for mutants in a hypothetical hormone
responsive pathway that activates transcription
Set up selection:
Hormone +
P
URA3
OFF
ON
P
lacZ
OFF
ON
2 strains of opposite mating type
MATa his3-11 ura3-52 Trp+ + reporters
MAT trp1-1 ura3-52 His+ + reporters
Selection for constitutives: Ura+ Blue
Selection for uninducibles: Ura- (5-FOAr) white
with hormone
Check Dominance/Recessiveness
Place recessives in complementation groups.
Dominants can be tested for allelism - linkage.
Epistasis
Clone, Sequence, Disrupt
Molecular + Biochemical Analysis
54
Genetic screens in yeast 3
A screen for dosage suppressors of a Ts mutant
Transform a genomic library on a 2 micron vector (50 copies per cell)
Plate transformants on selective media at the non-permissive
temperature of the Ts mutant
---- colonies come up after several days
Distinguish between plasmid dependent and
plasmid independent events.
Isolate plasmids and retest suppression.
Sequence, identify gene responsible for suppression.
55 Synthetic lethal screen
Red Color
Red Sectoring
Wild-type
Mutant(Ts)
Non-sectoring
Screen for mutagenized colonies that fail to lose
the color marked plasmid containing the wild-type gene.
Use strains of differing mating types
Demonstrate that the mutant phenotype is selecting for your gene and
not some other gene on the plasmid.
How?
Complementation grouping.
Cloning by complementation.
How do you know the gene you have isolated is actually the gene in which the
synthetic lethal mutation lies?
Two ways.