Transcript Forward Genetics

Phenotype
(Function)
Genetics
Forward Genetics
Gene A
Gene B
Gene C
P
Proteins
A
B
C
Survey
In classical or forward genetics, we commonly use
chemicals or radiation to generate mutations in model
organisms such as yeast, worm, fly or mouse.
In doing so, we typically try to generate mutations in
A: somatic cells
B: germline cells (gametes, sperm or oocytes)
C: not sure
Mutagenesis-sex chromosome
female
2n
Male
XX
XY
meiosis
Oocyte
gametes
Oocyte
X
Sperm
X
Y
50%
50%
X
fertilization
XX
50%
XY
50%
Mutagenesis-sex chromosome
female
2n
Male
Mutagen
XX
XY
meiosis
Oocyte
gametes
Oocyte
X
Sperm
X
x
fertilization
xX
xY
heterozygote
mutant
Y
Mutagenesis-autosome
female
2n
Male
A A
A A
meiosis
Oocyte
gametes
Oocyte
A
A
Sperm
x
A
A
50%
50%
fertilization
AA
50%
y
AA
50%
Mutagenesis-autosome
female
2n
X-ray
Male
A A
A A
meiosis
Oocyte
gametes
Oocyte
A
Sperm
x
y
A
A
50%
50%
A
some
a
fertilization
AA
50%
AA
50%
Aa
Heterzygous
mutant
Your opinions
When you mutagenize the gametes of P0 animals, you
usually do not get homozygous mutants in the F1
generation.
A: Yes
B: No
C: not sure.
Do you usually get homozygous mutants in the F2?
A: Yes
B: No
C: not sure.
F1 screens vs. F2 screens
Po WT
+
X
+
F1 mutant
m
+
Po WT
+
X
+
+
+
F1 WT
m
X
+
m
+
+
+
F2 mutant
m
m
But, how do we get the same m in two F1 and let them mate?
Balancer chromosomes
Chromosomes that suppress crossover. Homozygous of the
chromosome is either lethal or with a visible phenotype.
Usually contain inversions and translocations
A
B
wt
balancer
B
A
A
B
Resulting abnormal
chromosomes
B
A
Neither can be paired with WT
Traditional F2 screen in fly
X-ray
TM
*
X
TM
*
TM
X
F1
TM
*
*
X
*
*
F3 homozygotes
F2 screens in the worm
Hermaphrodite
Po
XX
meiosis
Sperm
gametes
Oocyte
X
X
100%
100%
x
fertilization
XX
100%
Self-progeny
xX
F1
Worm F2 screen
xX
F1
meiosis
Sperm
gametes
X
Oocyte
x
X
X
xx
F2
Homozygous mutant
1/4
summary
Po WT
+
X
+
+
+
Po WT
mutagen
F1 WT
m
X
+
+
+
F1 WT
m
X
+
m
+
F3 mutant
m
m
Fly, mouse, …
+
+
mutagen
F1 WT
m
+
F2 mutant
m
m
worm
Basic mutagenesis in the worm
Treat with
50mM EMS
for 4 hrs
F1
each F1 carries two
mutagenized
chromosomes
m/+ heterozygotes for
each mutation
Recovering for a
few hours
P0
F1 eggs
Several L4 or young
adult worms/plate.
Multiple large plates
F2 eggs
F2
Remove P0 parents
after laying ~100 eggs
Total genomes screened = 2X # of F1 animals x # of F1 plates
Remove F1 worms
after laying ~ 2000
eggs for 20 hrs
Worms with m/m
genotype for each
mutation are mixed with
m/+ and +/+ animals
mutant worms (m/m)
are individually picked
on to a fresh small
plate.
Question
If you plan to mutagenize and screen for a
mutation in a tumor suppressor gene that
may leads to tumorigenesis, would you do
F1 screen or F2 screen?
A: F1
B: F2
Genetic and physical map
Genetic map
nDf41
mDf4
1.8
map unit
Cloned genes
rme-2
sup-23
Non-cloned genes
evl-7
2.0
unc-5
him-3
unc-77 mor-2
fem-1
2.2
skn-1
sup-41
Physical map
YACs
cosmids
Named genes
eP14
rme-2unc-5
fem-1
skn-1
nhr-48
Genetic map and physical map will completely unified when every
gene has been mutated. A: yes. B: No. C: not sure.
Figure 8.15. Linkage analysis between two mutations.
+; dpy
+; +
+ dpy
unc; +
unc +
unc; +
unc +
dpy
+ +
dpy
X
A few wild type
A few Dumpy (Dup)
Pick several male progeny
unc
unc
dpy
+
X
Uncoordinate (Unc)
Wild type
50%
unc ;
+
+
+
unc +
+
+
Segregate no dumpy
progeny, discard
50%
Pick several wild-type
hermaphrodites
and place each to one pate
If the two genes are unlinked
unc ; dpy
+
+
If the two genes are linked
unc +
+ dpy
Segregate dumpy progeny,
continue mapping with the plates
dpy ; unc
+
+
Situation 1. The two
genes are unlinked
Wild type 9/16
Phenotypes
of worms
In the plate
genotypes
dpy ; unc
+
+
dpy ;
+
+
+
+
+
; unc
+
+
+
;
+
+
4/16
2/16
Unc 3/16
Dpy 3/16
dpy ; unc
unc
+
2/16
dpy ; unc
+
dpy
; unc
unc
1/16
dpy ;
dpy
+
+
A single plate
+
+
2/16
1/16
Pick 20 Unc animals
If ~ 2/3 of these Unc worm segregate Dpy-and-Unc animals,
non-linkage between the two genes is deduced.
2/16
1/16
Dpy-and-Unc 1/16
dpy ; unc
dpy unc
2/16
+ genes
+ dpy
Situation 2. unc
The two
are linked
on
one
of
the
six
chromosomes
unc +
+
dpy
unc +
+ dpy
unc dpy
A single plate
Self fertilizing
Wild type 1/2
Unc 1/4
Dpy 1/4
Dpy-and-Unc 0
Phenotypes
+
dpy
unc +
Non-recombinant
Genotypes
+
+
unc +
unc +
unc dpy
unc +
Rare recombinants
dpy
dpy
unc
unc
dpy
dpy
unc dpy
+
dpy
Pick 20 Unc animals
majority
unc
unc
+
+
linkage between the genes is indicated by
segregation of no Dpy animals from all or the
majority of Unc animals.
Rare
unc
unc
dpy
dpy
The frequency of rare recombinants that
segregate Dpy animals is correlated with the
genetic distance between the two genes.
Figure 8.16. Example of genetic three point mapping
phenotype
genotype
dpy
unc
mapping strain
Wild type hermaphrodite
Most of the gametes
egl
meiosis
Rare gametes from a recombination event
dpy
unc
egl
dpy
Sperms or eggs
unc
Sperms or eggs
egl
Progeny from combination
of the common gametes
Wild type
Unc and Dpy
Progeny from combination
between common gametes and
recombinant gametes
Self-fertilizing
unc + dpp
+ egl +
Dpy non-Unc
unc + dpp
+ egl dpy
Unc non-Dpy
unc + dpp
unc + +
unc + dpp
unc + dpy
Wild type
Egl
+ egl +
+ egl +
Egl
unc + +
+ egl +
+ egl dpy
+ egl +
dpy
unc
Recombination occurs
to the left of egl
egl
to the right of the egl
dpy
dpy
egl
unc egl
Recognizable
recombinants
Progeny with
Egl phenotype
unc
unc + dpp
+ + dpy
unc + dpp
+ egl dpy
unc + dpp
unc egl +
unc + dpp
unc + +
Dpy non-Unc
Unc non-Dpy
No
Yes
unc egl
dpy
Map position
a
b
Dpy non-Unc
Unc non-Dpy
a
=
b
Yes
No
# of recombinations occurred to the left of egl
# of recombinations occurred to the right of egl
Figure 8.17. An example of genetic mapping using SNPs
egl
unc
X
unc
egl
A C. elegans strain from Hawaii.
SNPs between this strain and the
Bristol strain have been determined.
Genetic mutant derived from the
strain from Bristol, England.
The egl mutation is being mapped.
egl
unc
X
X
X
*1 2* *3 *4 *5 *6
The hybrid strain. Stars indicate
SNPs in the region
Select Unc but non-Egl recombinants
B
A
egl
unc
* *2 *3 *4 *5 *6
unc
unc
unc
C
egl
unc
* * * *4 *5 *6
unc
egl
* * * * * *6
Determine SNP #4 for all recombinant
worms by sequencing or digestion.
Worms A and B have #4 SNP from the Hawaii strain
Determine SNP #5 and #6 for those that
have lost SNP#4 (worm C only)
Worm C has SNP #6 but not #5: the egl gene maps to the right of SNP#5
Common steps involved in cloning C. elegans genes defined by mutations.
Genetic mutation
SNP mapping
RNAi of candidate genes
Mapping using
marker mutations
Microinjection of cosmid/YAC clones
Injection of subclones
sequencing mutant DNA
What would be the flow chart for cloning in yeast?
Fly?
Human?
Which is the strongest evidence for claiming the
cloning of the gene defined by the mutation?
A. The transgene put back into the animal can rescue
the mutant phenotype.
B. You find a missense mutation in this gene by
sequencing.
C. Reducing the gene activity by RNAi mimics the
mutant phenotype.
D. The gene is expressed in the tissue with the mutant
phenotype.
Figure 8.19. Microinjection transformation in C. elegans.
DNA solution is injected to the distal arms of the gonad
Injection
Select F1 transgenic animals, most are unstable
F2 transgenic animals, stable lines
Three types of markers
unc-119(-) mutant
unc-119(+)
gene as a
marker
Wild type
Wild type
A dominant rol6 mutant gene
as a marker
Roller
Wild type
a strongly
expressed GFP
gene as a
marker
Green worm
Figure 8.21. The prevailing model for the mechanism of RNAi
dsRNA
Introduced into cells
Dicer
Bind to Dicer-RDE1enzyme complex
dsRNA is cut to ~22 nt siRNA
Incorporated into RISC nuclease complex
Multiple-protein
components of RISC
Unwinding siRNAs, activation of RISC
Target mRNA
AAAAAAAA
5’
Cleavage of target mRNA
Figure 8.22. RNAi methods in C. elegans.
A. Injecting ds RNA into intestine or gonad
dsRNA
intestine
Transfer to plates
Observe phenotype in progeny
B. Soak worms with dsRNA solution
Soak for 24 hours
Transfer to plates
Observe phenotype in progeny
C. Feed the worms a bacterial strain that expresses dsRNA
Feed worms
the bacterial
strain
Grow the bacterial
strain containing the
vector expressing
dsRNA
Observe phenotype in progeny