Download Lin-12(+)

Phenotype
(Function)
Genetics
Gene A
Gene B
Gene C
P
Proteins
A
B
C
Four-winged fruit fly
Mutations in ultrabithorax
regulatory region transform the
3rd thoracic segment into 2nd
one.
Edward B. Lewis
Question
Lewis’s homeotic mutations shows that there is an
correlation between mutant phenotype and gene
functions. Each gene is responsive for a specific
function, thus, generating mutations in individual genes
is sufficient to uncover gene functions in development.
A: Agree
B: not Agree
- Homeotic phenotype, pleiotropic phenotypes and no phenotypes
Gene number
Functional diversity
The genomes:
- the number of genes
- yeast
6,400
- rice
32,000
- worm
20,000
- fly
13,000
- Human 30,000
What is your reaction to this number:
A: There are too few genes
B: There are too many genes
C: Both
Mutations
Specific phenotypes
Malor problem #1
most genes act in multiple developmental processes,
making it difficult to isolate mutations for a specific
role.
Approaches to deal with it:
- Isolate conditional mutations
- Using sensitized genetic screens to isolate partial
loss-of-function or hyperactive mutations
- Genetic mosaic screens/tissue specific knockouts
Pleiotropy often prevents the observation of phenotypes
of null alleles in specific tissue or at specific stages.
Early phenotypes of a null allele masks the late phenotypes
null/+
Heterozygous
mother
null/null
Dead embryo
homozygous
progeny
rf/+
rf/rf
uncoordinated
Late phenotypes of a null allele may masks the early phenotypes
m/+
Heterozygous
mother
Maternal product
provides early
essential
functions
m/+
m/m
m/m
Elimination of
maternal activity by
creating germline
mosaics or by RNAi
homozygous
progeny
Larval lethal or sterile
Dead embryo
An F1 screen for lethal or sterile mutations.
EMS
+
+
Po
Po
20 plates
3 days
m
+
F1 clone
F1
20 plates
Dead embryo or
larvae, or sterile adults
Isolate 20 F1s from
each Po plate
1
2
…
F2
20
+
+
1/4
m
+
1/2
m
m
Pick WT siblings to
individual plates
F2
400 plates;
800 mutagenized
genomes
Screen for lethal or
sterile phenotypes
No longer see
lethal progeny
Discard
Continue to
see lethal
progeny.
Repeat the
step to keep
the strain
1/4
lethal screen in fly
X-ray
TM
*
X
TM
*
TM
X
F1
TM
*
*
X
*
*
F3 homozygotes, lethal
Maintaining lethal or sterile mutations
let
let
unc
dpy
Balancer
Select wild type progeny
let
let
unc
dpy
Keep the animals that
continue to segregate
Dpy and Unc progeny
Balancer
Recombination within
the balanced region is
suppressed
Maternal effect lethal gene
null/+
null/null
X
+/+
null/null
null/+
null/null
Dead embryos
A: Dead embryos
B: wild type
C: not
sure
Dead
embryos
Mutations
Specific phenotypes
Malor problem #2
A large % of genes have no obvious knockout phenotypes
Yeast: 40% genes
Worm: KOs of a large % have no obvious phenotypes
Mouse: >30% knockouts have no drastic phenotypes
Even for gene with mutant phenotypes, they have
other functions not manifested by the phenotypes
Vote
A: Genes with no robust knockout phenotypes have no
important biological functions.
B: Genes with no robust knockout phenotypes have less
important functions than those with robust phenotype.
C: Genes with no robust knockout phenotypes have just
as important functions.
Why are there genetic redundancies
associated with our genome?
The genomes use the strategy to increase the
resilience to mutational effects.
A: yes
B: no
We may discuss more at the end.
Redundancy provided by duplicated genes
Homologs: genes with common ancestry.
- ortholog = common ancestry and function
- paralogs: some kind of common ancestry (seen
in structure or sequence), but different functions,
the consequences of "parallel evolution"
Redundancy provided by duplicated genes
A
Same biochemical functions
Function
B
Yeast Histone H2B has two genes encoding essentially the same protein
Genotype
H2B 1(-); H2B2 (+)
H2B 1(+); H2B2 (-)
H2B 1(-); H2B2 (-)
Phenotype
wild type
wild type
dead
H2B is an essential
component of nucleosomes
Mouse Syne 1/2 genes
Genotype
syne1(-); syne2 (+)
syne1(+); syne2 (-)
syne1(-); syne2 (-)
Phenotype
wild type
wild type
die at birth,
Syne1
Syne12
Nuclear
membrane
functions
Redundancy provided by duplicated genes
Question:
or C.
eleganslin-12
Notchand
receptors
TheExample
differences
between
glp-1 functions reflect
A: the differences
LIN-12 and
GLP-1
protein
lin-12(-) between the
epidermal
tissue
defect
structures.
glp-1(-)
germline defects
lin-12(-) & glp-1(-) embryonic lethal
B: the differences in their expression pattern.
Genotype
lin-12(-); glp-1 (+)
lin-12(+);glp-1 (-)
lin-12(-); glp-1 (-)
Phenoytpe
phenotype A
phenotype B
phenotype C
LIN-12
LAG-1
Function A
GLP-1
LAG-1
Function B
LIN-12
LAG-1
GLP-1
Function C
Redundancy provided by duplicated genes
Experiment (Greenwald and Strul):
lin-12 promoter
glp-1 promoter
Genotype
lin-12(-); glp-1 (+)
lin-12(+);glp-1 (-)
lin-12(-); glp-1 (-)
glp-1 coding region
acts as lin-12
lin-12 coding region
acts as glp-1
Phenoytpe
phenotype A
phenotype B
phenotype C
LIN-12
LAG-1
Function A
GLP-1
LAG-1
Function B
LIN-12
LAG-1
GLP-1
Function C
"Redundancy" by structurally unrelated genes?
Question :
Majority of the genetic redundancy we observed (for
example the “no phenotype” situation with 40% of
the yeast genes) are due to functional redundancy
provided by duplicated genes
A: Yes
B: No
C: do not have a clue
Genetic redundancy due to protein activities
on different targets in the same pathway
C
D
A
B
Genotype
ark-1(lf); gap-1(+)
ark-1(+); gap-1(lf)
ark-1(lf); gap-1(lf)
EGF
(signal)
EFGR
ARK-1
Function
Phenoytpe
wild type
wild type
Multivulva (90%)
RAS
Vulval
induction
GAP-1
P. Sternberg lab
90
% Multivulva
40
29
0
0
Ark-1
RTK
Sli-1
0
Gap-1
0
Unc-101
GRB2
ARK-1 UNC-101 SLI-1
(CBL)
SOS
Ark-1
Sli-1
Ark-1
Gap-1
RAS
Ark-1
Unc-101
RAF
GAP-1
Sternberg lab
Discovery of synMuv genes
mutagen
Wild type
Multivulva
Genotype
Phenotype
lin-8(-)
lin-9(-)
lin-8(-) & lin-9(-)
wild type
wild type
Multivulva
Horvitz and Sulston 1980
Synthetic Muv phenotype define redundant genetic pathways
ClassA synMuv
genes (4)
ClassB synMuv
genes (20)
Vulval differentiation
Genotype
No
structural
similarity
between
genes
Phenotype
Class A
Class B
+
+
Wild type
Class A
Class B
+
Wild type
Class A
Class B
+
-
Wild type
Class A
Class B
-
Multivulva
Ferguson and Horvitz, 1989
Later papers
Mutations
Specific phenotypes
Malor problem #2
A large % of genes have no obvious knockout phenotypes
Methods to deal with it:
- Multiple knockouts
- Genetic screens in sensitized background
- Synthetic screens
The concept and usage of mosaic analysis
- What is the problem? Why do we need mosaic analyses?
- Germline mutations vs. somatic mutations
- Mosaic analysis in Drosophila
- Mosaic analysis in C. elegans
- Genetic mosaic screens in fly
- Mosaic analysis in mouse
About mosaic analysis
A genetic mosaic is an organism carrying
cells of different genotypes
Question:
A somatic mutation in our body leads to a mosaic
genotype regarding the gene containing the mutation.
A: agree. B. disagree. C. not sure.
About mosaic analysis
Most of the classical geneticists have been
doing germ-line mutagenesis. Therefore, all
cells in a given animal have the same
genotype (non-mosaic).
A: agree. B. disagree. C. not sure.
About mosaic analysis
The vast majority of cancers are caused by
somatic mutations.
A: agree. B. disagree. C. not sure.
What about other human diseases?
Multiple steps (multiple mutations) in cancer formation
A concept
Phenotypes from mutations in somatic cells in a specific
tissue are often different from phenotypes of animals that
contain the mutation in every cell.
A: Yes
B: No
C: not sure
Why do we need mosaic analyses?
1. Determine the site of gene action.
Q: Does expression pattern tells us a gene’s action site?
A: Yes, always.
B. Only sometimes.
C. Tells us essentially nothing about the action site.
The cell or cells in which a gene is expressed is not
necessarily where the gene expression is needed for a
specific function
A concept
Where the abnormality caused by a mutation in a
gene is seen is not necessarily where the
expression of the gene is needed for the function.
Genetic mosaics permit a correlation between
cellular genotype and cellular phenotype
Mosaic analysis can be used to determine
the site of gene action.
Mosaics
(Genotype of the
ced-3 gene)
+
-
Phenotype of the
middle cell
Wild type
(cell death)
+
+
+ +
Mutant
(cell survives)
conclusion
The gene being tested acts
(A) cell autonomously
(B) cell non-autonomously
in the middle cell for its function in programmed cell death.
Cell-nonautonomous
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Hunter and Wood, Cell 1990
Drawing from ergitol.com
2. Determine gene functions in specific tissues
A. Dealing with pleiotropic phenotypes
B. Studying maternal gene function
zygote
or fly
(+)
Somatic
Cells (+)
(-)
Germ
Cells (-)
Germ-line mosaic
(- )
All progeny are mutants
and there is no maternal
wild-type gene product
Maternal effect gene is expressed during oogenesis.
The entire cell lineage of a C. elegans hermaphrodite.
From HHMI bulletin
Identify a defect in a specific cell lineage
Zygote (-)
Zygote (+)
(+)
(-)
Zygote (+)
(-)
(+)
Zygote (+)
(+)
(+)
(-)
Dead lava
Dead lava
Live
The gene is
required in the
The gene is not
required in the
A: “red” lineage
B: “blue” lineage
A: “red” lineage
B” “blue” lineage
Dead lava
The gene is required
in the “red” lineage
Yochem et al. 1997 used this method to determine the site of the essential Ras
gene function.
Methods for generating genetic mosaics
1. Tissue, cell or nuclear transplantaiton
2. Chromosome loss
Mosaic analysis in C. elegans. Examples.
3. Mitotic recombination
- induced by radiation (Drosophila example)
- induced by site specific recombinases
(Drosophila, mice)
mom-2(lf)
MS
MS
Mom-2 gene function
Thorpe et al. 1997 Cell
Mom-2 acting site: EMS or P2?
Mosaics: issue transplantation
mom
EMS
MS
P2
E
mom(-) mom(+)
mom(+) mom(-)
MS
MS
MS
E
Where is the action site of this mom gene? A: EMS. B: P2.
Methods for generating genetic mosaics
1. Tissue, cell or nuclear transplantaiton
Example:
Wnt action site in C. elegans early embryo.
2. Chromosome loss
Mosaic analysis in C. elegans. Examples.
3. Mitotic recombination
- induced by radiation (Drosophila example)
- induced by site specific recombinases
(Drosophila, mice)
Example #1:
Determine the gene action site of Notch protein
Mosatic in C. elegans
Contain gene tested
Contains a visible marker
Normal chromosomes
Free duplication or
Exchromosomal array
WT
Z1.ppp
Z4.aaa
50%
AC
VU
50%
VU
AC
ablation
100%
AC
100%
AC
Lin-12 mutants
lf
gf
100%
100%
AC
AC
VU
VU
Greenwald et al. Cell, 1983
lin-12
mutants
lf
gf
100%
100%
AC
AC
VU
VU
lin-12: A: promoting VU. B: inhibit AC. C: either. D: neither.
lin-12
Does lin-12 act as (A) part of the signal, or (B) part of
receiving mechanism?
How do you determine whether lin-12 is a gene for the signal or receptor?
Mosaic analysis
Z1
Lin-12(-)
Z1.ppp
If
Z4
Lin-12(+)
Z4.aaa
AC
VU
A: Lin-12 is a receptor
VU
AC
B: Lin-12 is a signal
Real experiment
ncl-1(-) lin-12(-)
Chromosomal genotype
ncl-1(-) lin-12(-)
ncl-1(+) lin-12(+)
free duplication
(or extrachromosomal array)
Mitotic division
ncl-1(+)
lin-12(+)
ncl-1(-) enlarged nucleoli
lin-12(-)
Seydou and Greenwald, Cell 1989
Z4
Z1
Z1.aa
Duplication near lin-12
Z4.aaa
Z1.ppp
Lin-12
nuc-1
Nuc-1 mutation cause nucleolus bigger
Z4.pp
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Indicating: lin-12 encodes a protein that act on (A) the signaling or (B) reception side ?
(Adapted from the Hartwell Genetic book)
Methods for generating genetic mosaics
1. Tissue, cell or nuclear transplantaiton
Example:
Wnt action site in C. elegans early embryo.
2. Chromosome loss
Mosaic analysis in C. elegans. Examples.
3. Mitotic recombination
- induced by radiation (Drosohila example)
- induced by site specific recombinases (Drosophila, mice)
WT
sevenless
How do they know it is a receptor?
- Structurally similar to receptor tyrosine kinase
- Mosaic analysis determined that they act in R7
- The protein is expressed in the R7 membrane
Sev receptor
R8
?
Undifferentiated cell
Sev
R7 photoreceptor cell
R7 differentiation
Somatic Recombination
*
*
*
**
**
*
**
Mitotic recombination: generating mosaic in Drosophila
No recombination
A B
a b
A B
a b
A B
a b
With recombination
A B
A B
A B
a b
a b
a b
1. Rare. Occur in G2
2. Enhanced by x-ray radiation.
Sevenless acts cell autonomously
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
Sev receptor
Adopted from Hartwell et al, Genetics
R7 to be
Discovery of the
Bride of Sevenless
- 1988, BOSS was isolated by Larry Zipurski’s lab by
the similar method - failure to response to UV.
-It has exactly the same Sevenless phenotype
Mosaic analysis of Boss
X-ray
WWW+
W+
bossbossboss+
boss+
Mitotic cross over
W- bossW- boss-
white
boss-
W+ boss+
W+ boss+
red
Boss+
A: boss acts in R7
B: boss acts in R8
RT fate is induced by RTK activation
Boss signal
R8
Boss
Sev receptor
Undifferentiated cell
Sev
R7 photoreceptor cell
R7 differentiation
Methods for generating genetic mosaics
1. Tissue, cell or nuclear transplantation
Example:
Wnt action site in C. elegans early embryo.
2. Chromosome loss
Mosaic analysis in C. elegans. Examples.
3. Mitotic recombination
- induced by radiation (Drosohila example)
- induced by site specific recombinases
(Drosophila, mice)
Mosaic genetic screens
1. Why do we need it?
2. Drosophila vs. C. elegans
Screen in Drosophila:
- create mosaic mutants
- screen homozygous mutants after one cross
Principle:
promote recombination in somatic cells using
yeast FLP recombinase system.
FRT screen
Traditional F2 screen
X-ray
X-ray
TM
*
*
X
TM
*
P(FRT)
P(FRT)
TM
X
F1
TM
*
X
*
*
X
*
*
F3 homozygotes
Induction of mitotic
recombination at the
FRT site, e.g. HSdrive FLP in flies
*
*
FRT: target for yeast FLP recombinase
*
In some cells
S phase
*
*
Induction of mitotic
recombination at the
FRT site, e.g. HSdrive FLP in flies
*
*
Xu and Rubin, 1995
FRT: target for yeast FLP recombinase
Cell type specific gene knockouts using loxP-Cre recombination system
LoxP mouse
Cre mouse
X
Exon 1
Exon 2
LoxP
Exon 3
LoxP
Endogenous gene X with
With LoxP sites flanking exon2
Cell-type-specific
promoter
Cre
All cells carry cre transgene mouse is
heterozygous for gene X knockout
LoxP -Cre mouse: all cells carry one copy of loxPmodified gene X, one copy of gene X knockout, and cre
genes
Cells not expressing Cre
Cells expressing Cre
Other methods to create mosaic genotypes:
Tissue specific promoter driving RNAi
Tissue specific promoter driving antisense
Tissue specific promoter driving expression of wild type gene in mutant
Somatic transposon excision.
….
Jim Priess’s screen
Treated with mutagen
lin-2(lf)
lin-2(lf)
Po
“Bag of worms” (egg-laying defective)
With a maternal lethal mutation
Without maternal lethal mutation
lin-2(lf) ; mel
+
lin-2(lf)
lin-2(lf)
lin-2(lf)
F1
1/4
2/4
1/4
lin-2(lf)
lin-2(lf)
F2
“Bag of worms”
“Bag of worms”
lin-2(lf) ; mel
+
lin-2(lf)
Keep for retaining the
mutation
Worms contain F3 dead eggs
lin-2(lf) ; mel
mel
lin-2(lf)
No viable progeny, but a
maternal lethal mutation is
identified
Synthetic Hyperproliferation Model
Rb
transcription
Rb
Cyclin levels
stability
FZR-1 LIN-23
+
+
–
–
fzr-1
+
–
+
–
Relative Cyclin
levels
Very Low
Low
Low
High, phenotype